Methods for administration and formulations for the treatment of regional adipose tissue

ABSTRACT

Provided herein are pharmaceutical formulations, methods, and systems for treating regional fat deposits and fat-related conditions and indications. Methods comprise administering a pharmaceutical formulation comprising a long-acting beta-2 adrenergic receptor agonist, for example, salmeterol, suitable for subcutaneous administration. Formulations comprise a pharmaceutical formulation that is suitable for subcutaneous injection comprising: (a) a lipophilic long-acting selective beta-2 adrenergic receptor agonist, or a salt thereof; and (b) at least one subcutaneously acceptable inactive ingredient.

This application is a continuation of U.S. application Ser. No.12/788,190, filed May 26, 2010, which claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Application No. 61/181,627, filed May 27,2009, U.S. Provisional Application No. 61/251,624, filed Oct. 14, 2009,and of U.S. Provisional Application No. 61/289,972, filed Dec. 23, 2009,the entire contents of each of which are hereby incorporated byreference herein and made part of this specification.

BACKGROUND OF THE INVENTION

Excess body fat is a significant health care issue in modern societies.Chronic health conditions promoted by excess body fat include, e.g.,cardiovascular disease and diabetes mellitus type 2. Moreover, excessbody fat greatly undermines personal appearance and self image.

Accumulation of fat stores can occur unevenly in the body. For example,some persons may accumulate fat predominantly in visceral areas whileothers predominately in the subcutaneous tissue. Gender differences mayalso be apparent with women accumulating fat in the thighs and lateralbuttocks and males in the waist. Women may accumulate fatty deposits ofthe thighs, which have a rumpled or “peau-de-orange” appearance,resulting in a condition referred to as cellulite. Cellulite may berelated to skin architecture which allows subdermal fat herniation,sometimes referred to as adipose papillae. Other factors that may berelated to cellulite include altered and/or reduced connective tissueseptae, vascular and lymph changes that lead to fluid accumulation andinflammation. Fat tissue may also accumulate in the form of a fibrousfatty deposit known as a lipoma. Utilization of fat stores may occurunevenly. Persons who have lost substantial weight may still haveregional pockets of fat accumulation that are resistant to reductionunless unhealthy extremes of weight loss are achieved. Exercise mayaffect subcutaneous fat stores differently, with deeper tissuesresponding with lipolysis and superficial stores being more resistant.Cellulite may also still be present despite weight loss, and lipomas aretypically not affected by weight loss.

SUMMARY OF THE INVENTION

Described herein are subcutaneous and transcutaneous pharmaceuticalformulations and methods of treatment using the pharmaceuticalformulations for the treatment of regional adipose tissue. It has beendetermined that therapeutic effectiveness of a lipophilic long-actingselective beta-2 adrenergic receptor agonist and/or glucocorticosteroid,as evidenced for example by the change in circumference of a patient'swaist, is achieved when a relatively low dose is administered to apatient. For example, in clinical trials, it is been determined thatpatients that were administered the lowest dose produced the greatesttherapeutic response as measured by reduction in adipose tissue. It hasalso been determined that greater therapeutic effectiveness is achievedwith less frequent weekly administrations of a lipophilic long-actingbeta-2 adrenergic receptor agonist. For example, greater efficacy isprovided to a patient when an amount equal to or less than about 0.5 μgof a lipophilic long-acting selective beta-2 adrenergic receptor agonistis administered to a patient once per week when compared to situationswhere about 10 μg of a lipophilic long-acting selective beta-2adrenergic receptor agonist is administered to a patient twice per week.See, e.g., FIG. 4. It has been further determined that therapeuticefficacy of the formulations provided herein does not necessarilyrequire a reduction in weight of the patient or alteration in exerciseroutine by the patient, but rather the efficacy of the pharmaceuticalformulations and methods of treatment described herein is independent ofthese factors and influences. Accordingly, provided herein, in certainembodiments, are pharmaceutical formulations in optimal dosage amountsthat provide maximal therapeutic effect in a patient.

In one aspect, described herein are pharmaceutical formulations andcompositions that are suitable for subcutaneous injection comprising:(a) less than about 20 μg of an adipose tissue-reducing lipophiliclong-acting selective beta-2 adrenergic receptor agonist or a saltthereof; and (b) at least one subcutaneously acceptable inactiveingredient. In another aspect, provided herein is a method for providingcosmetic fat reduction of a human comprising subcutaneouslyadministering at or near the waist or abdomen a composition comprising:(a) less than about 20 μg of an adipose tissue-reducing lipophiliclong-acting selective beta-2 adrenergic receptor agonist or a saltthereof; and (b) at least one subcutaneously acceptable inactiveingredient. In yet another aspect, provided herein is a method forproviding a cosmetic waist or abdomen reduction of at least twocentimeters in a human, comprising subcutaneously administering at ornear the waist of the human a composition comprising (a) less than about20 μg of an adipose tissue-reducing lipophilic long-acting selectivebeta-2 adrenergic receptor agonist or a salt thereof; and at least onesubcutaneously acceptable inactive ingredient.

Also provided herein are pharmaceutical formulations for, in certainsituations, reducing regional fat deposits in a subject. Furtherprovided herein are pharmaceutical formulations that are formulated toprovide a single session dose of salmeterol xinafoate in an amount thatis about 5 ng to about 20 μg. In other embodiments, the formulationcomprises a single session dose of fluticasone propionate in an amountthat is between about 1 μg and about 300 μg. In still furtherembodiments, the formulation comprises a weekly dose of salmeterolxinafoate in an amount that is between about 5 ng to about 20 μg. Instill further embodiments, the formulation comprises a weekly dose offluticasone propionate in an amount that is between about 50 ng andabout 25 μg. Also provided herein is a formulation that comprises asub-dose of salmeterol xinafoate in an amount that is between about 1 ngto about 20 μg. In another embodiment, the formulation comprises asub-dose of fluticasone propionate in an amount that is between about 5ng to about 25 μg.

Also described herein is a method for reducing adipose tissue in apatient comprising subcutaneously administering a pharmaceuticalformulation suitable for subcutaneous injection comprising: (a) alipophilic long-acting selective beta-2 adrenergic receptor agonist orglucocorticosteroid, or a salt, optical isomer, racemate, solvate, orpolymorph thereof; and (b) at least one subcutaneously acceptableinactive ingredient.

Described herein is a method for reducing the circumference of apatient's abdomen comprising subcutaneously administering at or near thepatient's abdominal region a pharmaceutical formulation suitable forsubcutaneous injection comprising: (a) a lipophilic long-actingselective beta-2 adrenergic receptor agonist or glucocorticosteroid, ora salt, optical isomer, racemate, solvate, or polymorph thereof; and (b)at least one subcutaneously acceptable inactive ingredient. In aspecific embodiment, the circumference of the patient's abdomen isreduced by at least two centimeters as assessed by tape measure. In someembodiments, the two centimeter reduction in the patient's waist orabdomen is evident at about 4 to 8 weeks from the first day oftreatment. In further or additional embodiments, the patient experiencesa change in body weight during a treatment period of less than about 5%,less than about 3%, less than about 2%, less than about 1%, or less than0.5%.

Provided herein is a method for treating regional fat accumulationcomprising subcutaneously administering a pharmaceutical formulationsuitable for subcutaneous injection comprising: (a) a lipophiliclong-acting selective beta-2 adrenergic receptor agonist orglucocorticosteroid, or a salt, optical isomer, racemate, solvate, orpolymorph thereof; and (b) at least one subcutaneously acceptableinactive ingredient.

Described herein is a method for inducing lipolysis in adipose tissuecomprising subcutaneously administering a pharmaceutical formulationsuitable for subcutaneous injection comprising: (a) a lipophiliclong-acting selective beta-2 adrenergic receptor agonist orglucocorticosteroid, or a salt, optical isomer, racemate, solvate, orpolymorph thereof; and (b) at least one subcutaneously acceptableinactive ingredient.

Described herein is a pharmaceutical formulation comprising an activeingredient consisting essentially of an adipose tissue-reducing amountof a lipophilic long-acting selective beta-2 adrenergic receptor agonistor a salt, optical isomer, racemate, solvate, or polymorph thereof andat least one subcutaneously acceptable inactive ingredient. In oneembodiment, the lipophilic long-acting selective beta-2 adrenergicreceptor agonist selectively partitions into adipose tissue relative toplasma. In another embodiment, the lipophilic long-acting selectivebeta-2 adrenergic receptor agonist is salmeterol. In yet anotherembodiment, the salt of salmeterol is a xinafoate salt. In a furtherembodiment, the at least one subcutaneously acceptable inactiveingredient is selected from about 0.5 to about 40% polyethylene glycol.In yet a further embodiment, the at least one subcutaneously acceptableinactive ingredient is selected from about 0.1 to about 10% polysorbate.In one embodiment, the polysorbate is polysorbate 80.

In another aspect, provided herein is a pharmaceutical formulationcomprising an adipose tissue-reducing amount of salmeterol or a salt,optical isomer, racemate, solvate, or polymorph thereof and at least onesubcutaneously acceptable inactive ingredient, wherein the formulationprovides a mean plasma C_(max) equal to or less than about 300 pg/mLwhen administered subcutaneously. In one embodiment, salmeterolselectively partitions into adipose tissue relative to plasma. Inanother embodiment, the formulation further comprises aglucocorticosteroid or a salt or solvate thereof. In yet anotherembodiment, the glucocorticosteroid is fluticasone or a salt thereof. Ina further embodiment, the glucocorticosteroid is fluticasone propionate.

In yet a further embodiment, the formulations provide a mean fluticasonepropionate plasma C_(max) of about 1 to about 100 pg/mL. In someembodiments, the formulations provide a mean fluticasone propionateplasma C_(max) that is undetectable using conventional methodology. Inanother embodiment, salmeterol and fluticasone propionate areco-administered in a single subcutaneous formulation.

In another aspect, provided is a pharmaceutical formulation comprisingan adipose tissue-reducing amount of salmeterol or a salt, opticalisomer, racemate, solvate, or polymorph thereof and at least onesubcutaneously acceptable inactive ingredient, wherein the formulationprovides a salmeterol partition ratio of between about 0.01 to about 0.2when administered subcutaneously. In one embodiment, salmeterolselectively partitions into adipose tissue relative to plasma. Inanother embodiment, the salmeterol partition ratio is about 0.1.

In yet another aspect, provided is a method for reducing adipose tissuein a subject comprising subcutaneously administering to the subject apharmaceutical formulation comprising an active agent consistingessentially of an adipose tissue-reducing amount of a lipophiliclong-acting selective beta-2 adrenergic receptor agonist or a salt,optical isomer, racemate, solvate, or polymorph thereof and at least onesubcutaneously acceptable inactive ingredient. In one embodiment, thelipophilic long-acting selective beta-2 adrenergic receptor agonistselectively partitions into adipose tissue relative to plasma. Inanother embodiment, the lipophilic long-acting selective beta-2adrenergic receptor agonist is salmeterol. In yet another embodiment,the salt of the lipophilic long-acting selective beta-2 adrenergicagonist is a xinafoate salt. In a further embodiment, the pharmaceuticalformulation further comprises a glucocorticosteroid or a salt or solvatethereof. In yet a further embodiment, the glucocorticosteroid isfluticasone propionate. In another embodiment, the pharmaceuticalformulation provides a mean plasma fluticasone propionate C_(max) ofabout 100 pg/mL to levels that are undetectable using conventionalmethodology.

In yet another aspect is a method for treating regional fat accumulationcomprising subcutaneously administering to a regional fat accumulationarea a pharmaceutical formulation comprising an adipose tissue-reducingamount of salmeterol or a salt, optical isomer, racemate, solvate, orpolymorph thereof and at least one subcutaneously acceptable inactiveingredient, wherein the formulation provides a mean plasma C_(max) equalto or less than about 300 pg/mL (including C_(max) levels that areundetectable using conventional methodology). In one embodiment,salmeterol selectively partitions into adipose tissue relative toplasma. In another embodiment, the salt is a xinafoate salt. In yetanother embodiment, the formulation further comprises aglucocorticosteroid or a salt, optical isomer, racemate, solvate, orpolymorph thereof. In another embodiment, the formulation comprisesfluticasone or a salt thereof. In a further embodiment, is a method fortreating regional fat accumulation wherein the glucocorticosteroid isfluticasone propionate. In one embodiment, the formulation provides amean plasma fluticasone propionate C_(max) of about 100 pg/mL to levelsthat are undetectable using conventional methodology.

In one aspect provided herein is a method for reducing a regional fatdeposit in a subject in need thereof comprising administering to thesubject, a parenteral formulation consisting essentially of atherapeutically effective amount of at least one compound for reducingdesensitization of beta-adrenergic receptors and a long-acting beta-2adrenergic receptor agonist. In some embodiments, the parenteralformulation is administered by subcutaneous administration. In someembodiments, the at least one compound comprises a glucocorticosteroid.In another embodiment, the formulation comprises fluticasone or a saltthereof. In a further embodiment, is a method for treating regional fataccumulation wherein the at least one compound is fluticasonepropionate. In some embodiments, the therapeutically effective amount ofthe at least one compound is in a form suitable for subcutaneousadministration.

In some embodiments, the method further comprises administering, inaddition to the at least one compound for reducing desensitization ofbeta-adrenergic receptors, a therapeutically effective amount alipophilic long-acting beta-adrenergic agonist that is selective for thebeta-2 adrenergic receptor (e.g., salmeterol). In some embodiments, theat least one compound for reducing desensitization of beta-adrenergicreceptors, for example, fluticasone propionate, is administeredsubcutaneously prior to administration of the afore-describedcomposition comprising a therapeutically effective amount of alipophilic long-acting beta-adrenergic agonist.

In another aspect provided herein is a method for performingliposuction, comprising performing liposuction on a subject in needthereof that has been administered a pharmaceutical formulation suitablefor subcutaneous administration comprising a therapeutically effectiveamount of at least one compound for reducing desensitization ofbeta-adrenergic receptors and a therapeutically effective amount of anadipose tissue-reducing amount of a lipophilic long-actingbeta-adrenergic agonist that is selective for the beta-2 adrenergicreceptor. Conversely, in another aspect provided herein is a method forperforming liposuction, comprising performing liposuction on a subjectin need thereof followed by administration of a pharmaceuticalformulation suitable for subcutaneous administration comprising atherapeutically effective amount of at least one compound for reducingdesensitization of beta-adrenergic receptors and a therapeuticallyeffective amount of an adipose tissue-reducing amount of a lipophiliclong-acting beta-adrenergic agonist that is selective for the beta-2adrenergic receptor.

In a further aspect provided herein is a method for reducing a regionalfat deposit in a subject in need thereof comprising administering to thesubject a therapeutically effective amount of one or more adrenergicreceptor pathway-active compounds (e.g., a catecholamine, an alphaadrenergic antagonist, forskolin, aminophylline, analogs thereof, or anycombination thereof) and a therapeutically effective amount of at leastone compound for reducing beta-adrenergic receptor desensitization(e.g., fluticasone propionate) and a therapeutically effective amount ofan adipose tissue-reducing amount of a lipophilic long-actingbeta-adrenergic agonist that is selective for the beta-2 adrenergicreceptor (e.g., salmeterol xinafoate) in a formulation suitable forsubcutaneous administration. In some embodiments, the therapeuticallyeffective amount of the one or more adrenergic receptor pathway-activecompounds and the therapeutically effective amount of an adiposetissue-reducing amount of a lipophilic long-acting beta-adrenergicagonist that is selective for the beta-2 adrenergic receptor areco-administered in a formulation suitable for subcutaneousadministration. In another embodiment, the therapeutically effectiveamount of an adipose tissue-reducing amount of a lipophilic long-actingbeta-adrenergic agonist selectively partitions into adipose tissuerelative to plasma. In a further embodiment, is a formulation suitablefor subcutaneous administration comprising an adrenergic receptorpathway-active compound (e.g., a catecholamine, an alpha adrenergicantagonist, forskolin, aminophylline, analogs thereof, or anycombination thereof) and a long-acting beta-2 receptor agonist, such assalmeterol. In another embodiment is a formulation comprising an alphaadrenergic receptor antagonist and a long-acting beta-2 receptoragonist, suitable for subcutaneous administration.

In some embodiments, provided herein is a method of treating a patientcomprising administering the patient a formulation comprising a singlesession dose of salmeterol xinafoate that is administered to a patientin an amount that is between about 0.5 ng to about 20 μg during eachweek of a 4-8 week treatment period. In still further embodiments,provided herein is a method of treatment comprising the administrationof a formulation to a patient that comprises a single session dose offluticasone propionate in an amount that is between about 1 μg and about30 μg during each week of a 4-8 week treatment period.

In a further aspect provided herein is a method for treating a dermalcondition, such as for example, psoriasis, hypopigmentation, stria,wrinkles, rhytids, vitiligo, and atopic dermatitis, in a subject in needthereof, comprising administering to the subject a pharmaceuticalformulation comprising an active ingredient consisting essentially of anadipose tissue-reducing amount of a lipophilic long-actingbeta-adrenergic agonist; and a subcutaneously acceptable carrier orexcipient thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the embodiments described herein are set forth withparticularity in the appended claims. A better understanding of thefeatures and advantages presently described herein will be obtained byreference to the following detailed description that sets forthillustrative embodiments, in which the principles are utilized, and theaccompanying drawings of which:

FIG. 1 is illustrative of the plasma concentrations of salmeterol andformoterol following intravenous and subcutaneous administration toGottingen minipigs.

FIG. 2A is illustrative of the plasma concentrations of two differentconcentrations of salmeterol xinafoate as well as a combination ofsalmeterol xinafoate and fluticasone propionate following subcutaneousadministration to subjects.

FIG. 2B is illustrative of the plasma concentration of fluticasonepropionate and a combination of fluticasone propionate and salmeterolxinafoate following subcutaneous administration to subjects.

FIG. 3A is illustrative of the change in full waist circumference frombaseline over an 8 week period in human patients in the followingtreatment groups: (1) 0.5 μg of salmeterol and 1 μg of fluticasone onceper week for four weeks; (2) 5.0 μg of salmeterol and 1 μg offluticasone once per week for four weeks; and (3) 10 μg of salmeteroland 1 μg of fluticasone once per week for four weeks.

FIG. 3B is illustrative of the change in full waist circumference frombaseline in human patients after 8 weeks of treatment in the followingtreatment groups: (1) 0.5 μg of salmeterol and 1 μg of fluticasone onceper week for four weeks; (2) 5.0 μg of salmeterol and 1 μg offluticasone once per week for four weeks; and (3) 10 μg of salmeteroland 1 μg of fluticasone once per week for four weeks. FIGS. 3A and 3Bdemonstrate a dose-response effect with the treatment group receivingthe 0.5 μg of salmeterol and 1 μg of fluticasone once per week for fourweeks evidencing the greatest change in full waist circumference ofabout 3.5 cm.

FIG. 4 is illustrative of the change in full waist circumference (in cm)from baseline in patients enrolled in the study described in Example 3Bafter 8 weeks of treatment with salmeterol in combination withfluticasone in human patients in the following treatment groups (withthe indicated total weekly dose of salmeterol defined by dose andfrequency of injection): (1) 0.5 μg of salmeterol per week; (2) 1.0 μgof salmeterol per week; (3) 5.0 μg of salmeterol per week; (4) 10 μg ofsalmeterol per week; and (5) 20 μg of salmeterol per week. FIG. 4demonstrates a dose-therapeutic efficacy (based on change in waist orabdomen circumference) response curve based on a weekly dose ofsalmeterol.

FIG. 5A is illustrative of a comparison of the plasma concentration (inpg/mL) at day 1 and day 22 of salmeterol xinafoate in human patientsadministered 52 μg salmeterol xinafoate (in combination with 22 μgfluticasone propionate) once per week pursuant to the study describedherein in Example 3A with reference to the plasma levels of salmeterolfor the orally inhaled ADVAIR DISKUS® 500/50 drug product.

FIG. 5B is illustrative of the comparison of the plasma concentration(in pg/mL) at day 1 and day 22 of fluticasone propionate in humanpatients administered 22 μg fluticasone propionate (in combination with52 μg salmeterol xinafoate) once per week pursuant to the studydescribed herein in Example 3A with reference to the plasma levels offluticasone for the orally inhaled ADVAIR DISKUS® 500/50 drug product.FIGS. 5A and 5B demonstrate that the systemic exposure limits ofsalmeterol xinafoate and fluticasone propionate will not exceed thepharmacokinetic limits of the commercially available ADVAIR DISKUS®500/50 drug product. The increase in Cmax and AUC in the pharmacokineticprofiles depicted in FIGS. 5A and 5B is suggestive of tissue remodeling(resulting from a reduced amount of adipose tissue).

FIG. 6 is illustrative of the full waist circumference (in cm) for allpatients at baseline, after 5 weeks, and after 8 weeks pursuant to thestudy described in Example 3B. FIG. 8 demonstrates that the mean waistor abdomen circumference was reduced in the patients enrolled in thestudy described in Example 3B.

FIG. 7A is illustrative of the change in full waist circumference (incm) from baseline over an 8 week period (including at time points of 1week post-treatment and 4 weeks post-treatment) pursuant to the studydescribed in Example 3B for patients in each of the following groups:(1) 0.5 μg of salmeterol and 1 μg of fluticasone twice per week for fourweeks; (2) 5.0 μg of salmeterol and 1 μg of fluticasone twice per weekfor four weeks; and (3) 10 μg of salmeterol and 1 μg of fluticasonetwice per week for four weeks.

FIG. 7B is illustrative of the change in full waist circumference (incm) from baseline after 8 weeks from the first day of treatment pursuantto the study described in Example 3B for patients in each of thefollowing groups: (1) 0.5 μg of salmeterol and 1 μg of fluticasone twiceper week for four weeks; (2) 5.0 μg of salmeterol and 1 μg offluticasone twice per week for four weeks; and (3) 10 μg of salmeteroland 1 μg of fluticasone twice per week for four weeks. FIGS. 7A and 7Bdemonstrate a dose-related effect whereby the treatment group thatreceived the 0.5 μg of salmeterol and 1 μg of fluticasone twice per weekfor four weeks evidenced the greatest change of about 2.9 cm withrespect to patients receiving two injections per week.

FIG. 8 is illustrative of the mean body weight (in kg) at baseline, 4weeks from start of treatment, 1 week post-treatment, and 4 weekspost-treatment, for all patients enrolled in the study described inExample 3B. FIG. 8 demonstrates that the patients enrolled in the studydescribed in Example 3B did not evidence a significant change in weightduring 8 weeks of study.

DETAILED DESCRIPTION OF THE INVENTION

Adipose tissue is the primary energy storage tissue of the body. Fatcells, or adipocytes, store this energy in the form of triglycerides.Triglycerides are mobilized from fat stores to provide caloric energy tothe body through hormonal induction of triglyceride hydrolysis. Thisprocess releases free or non-esterified fatty acids and glycerol intothe blood for use by other body tissues. The breakdown of triglyceridesfrom fat store is referred to as lipolysis. Growth of new adipocytesalso occurs, which is referred to as adipogenesis. One of the primaryneurotransmitters that control lipolysis in the body are thecatecholamines epinephrine and norepinephrine. Adipose tissue hasbeta-1, 2, and 3 adrenergic receptors and alpha-2 adrenergic receptors.Binding of beta-adrenergic receptor agonists (“beta-adrenergicagonists”) to beta-adrenergic (“beta”) receptors in adipose tissueresults in adipocyte lipolysis. Beta-adrenergic receptor activation alsoinhibits adipogenesis. In humans, beta-2 receptors are the most abundanton fat cell surfaces and the primary mediator of beta-adrenergicreceptor-stimulated lipolysis. Stimulation of lipolysis bybeta-adrenergic agonists is mediated by adenylate cyclase and increasedformation of cyclic adenosine monophosphate (cyclic AMP, cAMP).

Long-acting beta-2 adrenergic receptor agonists, such as salmeterol andformoterol, reduce regional fat deposits or adipose tissue regions bybinding to beta receptors, resulting in adipocyte lipolysis. The use oflong-acting beta-2 adrenergic receptor agonists, however, carries withit possible side effects that are potentially life-threatening. Forexample, use of long-acting beta-2 adrenergic receptor agonists mayresult in cardiovascular problems such as angina, hypertension orhypotension, tachycardia, palpitations, and arrhythmias. Thus, whilelong-acting beta-2 adrenergic receptor agonists may reduce regional fatdeposits and adipose tissue regions they may also cause increased heartrate and palpitations.

It has been found that certain lipophilic long-acting selective beta-2adrenergic agonists administered subcutaneously in appropriate amountsoptionally with an appropriate amount of a certain glucocorticosteroidreduces regional fat deposits with limited systemic exposure compared toother long-acting beta-2 adrenergic agonists. One possible reason forthis result is that the lipophilic nature of certain long-acting beta-2adrenergic receptor agonists allows selective partitioning into theadipose tissue relative to plasma. The lipophilicity of certainlong-acting beta-2 adrenergic receptor agonist contributes, in part, toproviding relatively low levels of the agonist systemically. Combinedwith appropriately administered amounts via subcutaneous injection,certain lipophilic long-acting beta-2 adrenergic receptor agonists mayprovide therapeutic effectiveness in reducing regional fat depositsand/or adipose tissue with a reduced risk of producing cardiovascularside effects.

It has also been determined that of the doses of the formulationsprovided herein containing a lipophilic long-acting selective beta-2adrenergic receptor agonist that have been administered to humanpatients, the lowest dose was the most effective. It has also beendetermined that greater therapeutic effectiveness is achieved with lessfrequent weekly administrations of a lipophilic long-acting beta-2adrenergic receptor agonist. For example, it is been determined thatgreater efficacy is provided to a patient when about 10 μg of alipophilic long-acting selective beta-2 adrenergic receptor agonist isadministered to a patient once per week (and even greater therapeuticeffect with administration of less than about 10 μg, for example anamount that is less than or equal to about 0.5 μg, when administered tothe patient once per week) when compared to a dose of about 10 μg of alipophilic long-acting selective beta-2 adrenergic receptor agonist thatis administered twice per week. It has also been found that therapeuticefficacy, for example, as measured by a decrease in waist or abdomencircumference in patients administered a lipophilic long-actingselective beta-2 adrenergic receptor agonist and/or glucocorticosteroiddoes not necessarily require a reduction in weight or alteration inexercise routine of the patient.

GLOSSARY OF CERTAIN TERMINOLOGY

A “therapeutically effective amount,” as used herein, refers to asufficient amount of an agent (e.g., a long-acting beta-2 agonist) or acompound being administered which will relieve to some extent one ormore of the symptoms of the disease or condition being treated. Theresult can be reduction and/or alleviation of the signs, symptoms, orcauses of a disease, or any other desired alteration of a biologicalsystem. For example, an “effective amount” for therapeutic uses is theamount of the composition including a compound as disclosed hereinrequired to provide a clinically significant decrease in diseasesymptoms without undue adverse side effects. An appropriate “effectiveamount” in any individual case can be determined using techniques, suchas a dose escalation study. The term “therapeutically effective amount”includes, for example, a prophylactically effective amount. An“effective amount” of a compound disclosed herein, such as a selectivebeta-2 agonist used alone or in combination with other compounds (e.g.,a compound for reducing beta-2 adrenergic receptor desensitization), isan amount effective to achieve a desired pharmacologic effect ortherapeutic improvement without undue adverse side effects. It is to beunderstood that “an effect amount” or “a therapeutically effectiveamount” can vary from subject to subject, due to variation in metabolismof beta-2 agonists and compounds used in combination with beta-2agonists (e.g., glucocorticosteroids), age, weight, general condition ofthe subject, the condition being treated, the severity of the conditionbeing treated, and the judgment of the prescribing physician.

As used herein an “adipose tissue-reducing” amount refers to asufficient amount of the lipophilic long-acting beta-2 adrenergicreceptor agonist needed to reduce adipose tissue. It is to be understoodthat the amount sufficient to decrease the adipose tissue will vary fromsubject to subject due to variation in metabolism of the lipophiliclong-acting beta-2 adrenergic receptor agonist, with age, weight,general condition of the subject, the severity of the condition beingtreated, and the judgment of the prescribing physician.

As described herein a “reduced or minimized risk of producingcardiovascular side effects” amount refers to an amount of thelipophilic long-acting beta-2 adrenergic receptor agonist used whichdoes not result in clinically significant cardiovascular side effects.It is to be understood that the amount used will vary from subject tosubject due to variation in metabolism of the lipophilic long-actingbeta-2 adrenergic receptor agonist, with age, weight, general conditionof the subject, the severity of the condition being treated, and thejudgment of the prescribing physician.

“Plasma concentration” refers to the concentration of a substance suchas a therapeutic agent, in blood plasma or blood plasma of a subject. Itis understood that the plasma concentration of a therapeutic agent mayvary many-fold between subjects, due to variability with respect tometabolism of therapeutic agents. In accordance with one aspect, theplasma concentration of a long-acting beta-2 adrenergic receptor agonistor a glucocorticosteroid varies from subject to subject. Likewise, insome embodiments, values such as maximum plasma concentration (C_(max))or time to reach maximum plasma concentration (T_(max)), or total areaunder the plasma concentration time curve (AUC) varies from subject tosubject. Due to this variability, the amount necessary to constitute “atherapeutically effective amount” of a lipophilic long-acting beta-2adrenergic receptor agonist and/or a glucocorticosteroid is varies fromsubject to subject. It is understood that in some embodiments, when meanplasma concentrations are disclosed for a population of subjects, thesemean values include substantial variation.

“Pharmacodynamics” refers to the factors that determine the biologicresponse observed relative to the concentration of drug at a site ofaction.

“Pharmacokinetics” refers to the factors that determine the attainmentand maintenance of the appropriate concentration of drug at a site ofaction.

A “measurable plasma concentration” or “measurable plasma concentration”describes the blood plasma or blood plasma concentration, typicallymeasured in mg, μg, or ng of therapeutic agent per mL, dL, or L of bloodplasma, of a therapeutic agent that is absorbed into the bloodstreamafter administration. One in the field would be able to measure theplasma concentration or plasma concentration of a lipophilic long-actingbeta-2 adrenergic receptor agonist or a glucocorticosteroid.

As used herein, the term “co-administered,” refers to the administrationof two or more therapeutic agents in a single formulation or separateformulations or routes of administration in any order for the purpose oftreating the same health condition (e.g., a lipoma) in the same subject.

Some embodiments comprise optically pure isomers of the lipophilicbeta-adrenergic agonist(s), which improve lipolysis and adipogenesisinhibition and reduce the risk of producing potential cardiovascularside effects. In some embodiments, these optically pure isomers allowformulations comprising larger loadings of an active ingredient, forexample, by eliminating one or more isomers with no physiologicaleffect, a lesser a physiological effect, a negative effect, and/or anundermined physiological effect. Removing the undesired bounds of aracemic mixture isolates the active isomer, or eutomer, thereby allowingmore eutomer to be loaded in a give formulation by removing the inactivecomponents.

Two stereogenic centers in a molecule generally generate twodiastereomers, referred to herein as (R*,R*) and (R*,S*), and theirenantiomers. Diastereomers are stereoisomers that are not enantiomers,that is, the mirror image of one diastereomer is not superimposable onanother diastereomer. Enantiomers are stereoisomers that are mirrorimages of each other. A racemate is a 1:1 mixture of enantiomers. Theenantiomers of the (R*,R*) diastereomers are referred to as the (R,R)and (S,S) enantiomers, which are mirror images of each other andtherefore share some chemical and physical properties, for examplemelting points. Similarly, the (R,S) and (S,R) isomers are enantiomersof the (R*,S*) enantiomer. For example, some embodiments compriseoptically pure isomers of other lipophilic beta-2 agonists, for example,(R)-salmeterol.

Additionally, in some embodiments, a lipophilic, long-acting selectivebeta-2 agonists is lipophilic, thereby providing a pharmaceuticalformulation with activity in fat tissue. In some embodiments, thelipophilic agonist is salmeterol. In further embodiments, thelipophilicity of salmeterol provides prolonged exposure to the adiposetissue. In some embodiments, the agent is not salmeterol, but has asimilar lipophilicity to salmeterol.

Salmeterol has high lipid solubility, compared to other long-actingbeta-2 adrenergic receptor agonists, such as for example, formoterol,which extends its residence time in the adipose tissue and/or in one ormore adipose cells. Some embodiments of the subcutaneous formulationcomprise a highly lipophilic beta-adrenergic agonist, which reduces oreliminates the need for a sustained or controlled release carrier due topartitioning and sequestration in the adipose tissue thereby prolongingthe treatment effect. In some embodiments, lipophilic beta-adrenergicagonists with an oil-water partition coefficient of at least about 1000or at least about 10,000 to 1 are used. For example, salmeterol is atleast 10,000 times more lipophilic than albuterol, a short-actinghydrophilic beta-adrenergic agonist.

A “treatment period” is defined as the period of time the patient isunder a physician's care or direction, which may vary from patient topatient, and may be dependent on metabolism of the lipophiliclong-acting beta-2 adrenergic receptor agonist, glucocorticosteroid,and/or other active ingredient administered to the patient, age, weight,general condition of the subject, the severity of the condition beingtreated, and the judgment of the prescribing physician. In someembodiments, the treatment period comprises between 1 week and 52 weeks,longer than 52 weeks, or any week in between 1 and 52.

A “weekly dose” is the total amount of active ingredient administered toa patient during a single week. For example, in situations with morethan a single administration occurs during a week, the weekly dose isthe total amount of active ingredient provided to the patient in eachadministration that occurs during the week.

A “periodic dose” is the frequency at which a dose is administered to apatient during a period.

A “single session dose” is the total amount of active ingredientadministered to a patient during a single visit for treatment by ahealthcare professional or, in situations of self-administration, asingle session dose is the total amount of active ingredientadministered to the patient by self-administration in a single session.

In some embodiments, a single session dose is divided into smalleramounts and administered to a patient in one or more “sub-doses.” Insome embodiments, each “sub-dose” is subcutaneously delivered to apatient by injection, e.g., using a syringe or is administered to thepatient transcutaneously.

The phrases “patient” and “subject” are used interchangeably herein. Insome embodiments, the patient or subject is a human. In further oradditional embodiments, the patient or subject is an animal. In someembodiments, the animal is a human, a common household pet, includingfor example a cat or a dog, or a species of the animal kingdom. In someembodiments, the patient is a non-murine animal.

Active Ingredients

In one aspect, provided herein are pharmaceutical formulations suitablefor subcutaneous or transcutaneous administration and methods oftreatment comprising subcutaneously or transcutaneously administering toa patient a pharmaceutical formulation (including all of the methods oftreatment described herein) wherein the pharmaceutical formulationcomprises: (a) an adipose tissue-reducing amount of a lipophiliclong-acting selective beta-2 adrenergic receptor agonist orglucocorticosteroid, or a salt, optical isomer, racemate, solvate, orpolymorph thereof; and (b) at least one subcutaneously ortranscutaneously acceptable inactive ingredient. In some embodiments,provided herein are pharmaceutical formulations, and methods oftreatment, comprising administration of the pharmaceutical formulationsto a patient, wherein the formulation is suitable for subcutaneousadministration. In further or additional embodiments, the pharmaceuticalformulation is suitable for transcutaneous administration.

In some embodiments, the pharmaceutical formulation comprises alipophilic long-acting selective beta-2 adrenergic receptor agonist, ora salt, optical isomer, racemate, solvate, or polymorph thereof and aglucocorticosteroid, or a salt, optical isomer, racemate, solvate, orpolymorph thereof. For example, in some embodiments, the lipophiliclong-acting selective beta-2 adrenergic receptor agonist is salmeterolxinafoate and the glucocorticosteroid is fluticasone propionate.

In other embodiments, the pharmaceutical formulation consistsessentially of a lipophilic long-acting selective beta-2 adrenergicreceptor agonist, or a salt, optical isomer, racemate, solvate, orpolymorph thereof. In still further embodiments, the pharmaceuticalformulation consists essentially of a glucocorticosteroid, or a salt,optical isomer, racemate, solvate, or polymorph thereof. In additionalembodiments, the pharmaceutical formulation consists essentially of alipophilic long-acting selective beta-2 adrenergic receptor agonistand/or glucocorticosteroid, or a salt, optical isomer, racemate,solvate, or polymorph thereof.

Beta-2 Adrenergic Receptor Agonists

In one aspect, provided herein are pharmaceutical formulations suitablefor subcutaneous or transcutaneous administration comprising alipophilic long-acting selective beta-2 adrenergic receptor agonist,including, for example, salmeterol or2-(hydroxymethyl)-4-{1-hydroxy-2-[6-(4-phenylbutoxyl)hexylamino]ethyl}phenol, or its salts, optical isomers, racemates,solvates or polymorphs thereof and when used in the appropriate amountsand administered transcutaneously or subcutaneously, provides atherapeutic effect for reducing regional fat deposits and/or adiposetissue with limited systemic exposure, and consequently, a reduced riskof producing cardiovascular side effects. In one embodiment is asubcutaneous or transcutaneous preparation for the reduction of adiposetissue and/or the reduction in regional fat deposits comprising anadipose tissue-reducing amount of a lipophilic long-acting selectivebeta-2 adrenergic agonist wherein the formulation does not result inhigh systemic levels when administered subcutaneously ortranscutaneously. In another embodiment, the lipophilic long-actingselective beta-2 adrenergic receptor agonist is salmeterol. In yetanother embodiment, the agonist is a polymorph of salmeterol, such asfor example, polymorph I and II. Such subcutaneous or transcutaneouspreparations provide the required tissue concentration of salmeterolneeded to reduce adipose tissue and/or reduce regional fat deposits witha minimized or reduced risk of producing the side effects typicallyassociated with the administration of beta-2 adrenergic receptoragonists, including other long-acting beta-2 adrenergic receptoragonists. Additionally, the use of salmeterol in a subcutaneous ortranscutaneous preparation provides therapeutically effective dosageswithout producing relatively high systemic levels found when using otherlong-acting beta-2 adrenergic receptor agonists, such as for example,formoterol.

In another aspect, provided herein are pharmaceutical formulationssuitable for subcutaneous or transcutaneous administration and methodsof treatment comprising subcutaneously or transcutaneously administeringto a patient a pharmaceutical formulation (including all of the methodsof treatment described herein) wherein the pharmaceutical formulationcomprises a beta-2 adrenergic receptor agonist.

In some embodiments, provided herein are pharmaceutical formulations andmethods of treatment comprising a beta-2 adrenergic receptor agonist, ora salt, optical isomer, racemate, solvate, or polymorph thereof, that ischaracterized by at least one of the following properties: lipophilic;selective for the beta-2 adrenergic receptor; and long-acting. In someembodiments, the beta-2 adrenergic receptor agonist is selective for thebeta-2 adrenergic receptor. In further or additional embodiments, thebeta-2 adrenergic receptor agonist is lipophilic. In further oradditional embodiments, the beta-2 adrenergic receptor agonist islong-acting.

In some embodiments, the beta-2 adrenergic receptor agonist isbambuterol, bitolterol, broxaterol, carbuterol, carmoterol, clenbuterol,ibuterol, sulfonterol, isoproterenol, trimetoquinol, formoterol,desformoterol, hexoprenaline, ibuterol, indacaterol, isoetharine,isoprenaline, isoproterenol, levalbuterol, metaproterenol, picumeterol,pirbuterol, procaterol, reproterol, rimiterol, salbutamol, salmeterol;sulfonterol, terbutaline, trimetoquinol, tulobuterol, TA-2005(8-hydroxy-5-((1R)-1-hydroxy-2-(N-((1R)-2-(4-methoxyphenyl)-1-methylethyl-)amino)ethyl)-carbostyrilhydrochloride), QAB-149 (Novartis), TA-2005, GSK-159797, or GSK-642444,or a salt, optical isomer, racemate, solvate, or polymorph thereof.

In some embodiments, the beta-2 adrenergic receptor agonist islong-acting and is selected from salmeterol, formoterol, bambuterol, orclenbuterol. In further or additional embodiments, the beta-2 adrenergicreceptor agonist is ultra long-acting. In some embodiments, the ultralong-acting beta-2 adrenergic receptor agonist is selected fromindacaterol, carmoterol, QAB-149, CHF-4226, TA-2005, GSK-159797, andGSK-642444.

In some embodiments, provided herein are pharmaceutical formulationscomprising an active ingredient consisting essentially of an adiposetissue-reducing amount of a lipophilic long-acting selective beta-2adrenergic receptor agonist or a salt, optical isomer, racemate,solvate, or polymorph thereof and at least one subcutaneously ortranscutaneously acceptable inactive ingredient. In one embodiment, thelipophilic long-acting selective beta-2 adrenergic receptor agonistselectively partitions into adipose tissue relative to plasma.

In another embodiment, the lipophilic long-acting selective beta-2adrenergic receptor agonist is salmeterol(±2-(hydroxymethyl)-4-[1-hydroxy-2-[6-(4-phenylbutoxyl)hexylamino]ethyl]-phenol,CAS Reg. No. 94749-08-3, shown below as compound 1).

In other embodiments, the lipophilic, long-acting, selective beta-2agonist is a polymorph of salmeterol. In a further embodiment, thepolymorph is polymorph I or II. In further embodiments, the formulationuses a mixture of salmeterol polymorphs. In yet another embodiment, thesalt of the lipophilic long-acting selective beta-2 adrenergic receptoragonist is a xinafoate salt. In some embodiments, the lipophiliclong-acting selective beta-2 adrenergic receptor agonist is salmeterolxinafoate.

Some embodiments provide adrenergic modulation through the use ofpharmaceutical compositions comprising an adipose tissue-reducing andlipophilic, long-acting selective beta-2 receptor agonist activeingredient administered subcutaneously or transcutaneously, either alone(as single agent therapy), or in combination with at least a secondactive ingredient (as a combination therapy). Thus, in some embodiments,the pharmaceutical formulations (and corresponding methods of treatmentprovided herein) consist essentially of a lipophilic, long-acting,selective beta-2 agonist, for example, salmeterol, physiologicallyacceptable salts, optical isomers, racemates, solvates, polymorphs, orcombinations thereof, wherein the formulation is suitable forsubcutaneous or transcutaneous administration. In further or additionalembodiments, the pharmaceutical formulation comprises a lipophilic,long-acting, selective beta-2 agonist, for example, salmeterol,physiologically acceptable salts, optical isomers, racemates, solvates,polymorphs, or combinations thereof, and at least a second activeingredient, wherein the formulation is suitable for subcutaneous ortranscutaneous administration.

Lipophilic, long-acting, selective beta-2 agonists, for example,salmeterol are used in some embodiments. In other embodiments, salts,optical isomers, racemates, polymorphs, and/or solvates of beta-2agonists have the desired activity and are accordingly provided forherein. Unless otherwise specified, references to an active ingredient,for example, to salmeterol, include the compound itself as well as aphysiologically acceptable analogs, salts, optical isomers, racemates,polymorphs, solvates, or combinations thereof.

In some embodiments, salmeterol is used in the compositions and methodsdescribed herein. Depending on the tissue, salmeterol may exhibitpartial agonist activity, which is believed to reduce receptordesensitization and may limit arrestin signaling leading to lessreceptor down-regulation. In some embodiments, salmeterol is present asa physiologically acceptable salt, optical isomer, racemate, solvate,and/or polymorph thereof. Suitable physiologically acceptable salts ofsalmeterol include, but are not limited to acid addition salts derivedfrom inorganic and organic acids, such as the hydrochloride,hydrobromide, sulfate, phosphate, maleate, tartrate, citrate, benzoate,4-methoxybenzoate, 2-hydroxybenzoate, 4-hydroxybenzoate,4-chlorobenzoate, p-toluenesulphonate, methanesulphonate, ascorbate,salicylate, acetate, fumarate, succinate, lactate, glutarate, gluconate,tricarballylate, hydroxynaphthalenecarboxylate,1-hydroxy-2-naphthalenecarboxylate, 3-hydroxy-2-naphthalenecarboxylate,oleate, combinations thereof, and the like. In some embodiments,salmeterol is provided as the 1-hydroxy-2-naphthalene carboxylate salt(hydroxynaphthoate, also known as xinafoate).

Agents for Reducing or Preventing Desensitization

Beta-adrenergic activity is enhanced by preventing or reducingdesensitization (tachyphylaxis) that can occur with continuous exposureof adipocytes to adrenergic agonists as discussed above. “Compounds thatreduce desensitization of beta-adrenergic receptors” (e.g., reducedesensitization of a target tissue to a beta-adrenergic agonist) includeall suitable compounds that reduce tolerance of the target tissue to thebeta-adrenergic receptor agonists, including glucocorticosteroids andsuitable antihistamines, for example, ketotifen, and thyroid hormones,for example T3 and T4.

Glucocorticosteroids are also referred herein as “anti-inflammatorysteroids,” “glucocorticosteroids,” and/or “corticosteroids.”Glucocorticosteroids are believed to sensitize regional fataccumulations by increasing the number of surface beta-2 receptors,thereby favoring lipolysis or fat reduction over fat storage. It is alsounderstood that glucocorticosteroids also decrease the number of alpha-2receptors. Glucocorticosteroids also stabilize or reduce receptordown-regulation especially when given simultaneously with abeta-adrenergic agonist. Of note, estrogen can induce the expression ofalpha-2 adrenergic receptors in subcutaneous adipose tissue in womenresulting in a ratio of beta-2 receptor to alpha-2 receptor of lessthan 1. Thus, in one embodiment is a pharmaceutical compositioncomprising a beta-2 receptor agonist in combination with an alpha-2antagonist and a pharmaceutically acceptable excipient. In oneembodiment, the composition is suitable for subcutaneous ortranscutaneous administration.

Glucocorticosteroids

Some embodiments of the composition comprising one or moreglucocorticosteroids are effective in treating regions of fat comprisinga reduced number of beta-2 receptors and or an increased number ofalpha-2 receptors, which are resistant to beta-adrenergic stimulation oflipolysis or inhibition of adipogenesis, for example, subcutaneousadipose tissue, especially in women.

Without wishing to be bound by theory, it is believed thatglucocorticosteroids or other compounds for reducing desensitization ofbeta-adrenergic receptors increase lipolysis, adipogenesis inhibition,and/or regional fat reduction during beta-adrenergic agonist exposure.Thus, in some embodiments, a therapeutically effective amount of acompound (e.g., a glucocorticosteroid) for reducing desensitization ofbeta-adrenergic receptors is administered to increase lipolytic activityand/or increase the number of beta-receptors in the target tissue, andthereby increase fat deposit reduction. In some embodiments, a patientis administered a pharmaceutical formulation suitable for subcutaneousor transcutaneous administration comprising a therapeutically effectiveamount of a long-acting beta-2 adrenergic receptor agonist and aglucocorticosteroid. For example, the compound for reducingdesensitization of beta-adrenergic receptors is formulated suitable forsubcutaneous administration. In further or additional embodiments, thecompound for reducing desensitization of beta-adrenergic receptors isformulated suitable for transcutaneous administration. In someembodiments, the pharmaceutical formulation further comprises atherapeutically effective amount of a lipophilic selective beta-2adrenergic agonist that is selective for the beta-2 adrenergic receptor(e.g., salmeterol). In some embodiments, the lipophilic selectivebeta-adrenergic agonist is formulated as a subcutaneous ortranscutaneous formulation.

In some embodiments, a compound for reducing desensitization ofbeta-adrenergic receptors is a glucocorticosteroid. Thus, in certainembodiments, provided herein are pharmaceutical formulations and methodsof treatment comprising subcutaneously or transcutaneously administeringto a patient a pharmaceutical formulation (including all of the methodsof treatment described herein) wherein the pharmaceutical formulationcomprises: (a) an adipose tissue-reducing amount of a lipophiliclong-acting selective beta-2 adrenergic receptor agonist orglucocorticosteroid, or a salt, optical isomer, racemate, solvate, orpolymorph thereof; and (b) at least one subcutaneously ortranscutaneously acceptable inactive ingredient. In some embodiments,provided herein are pharmaceutical formulations, and correspondingmethods of treatment, comprising administration of the pharmaceuticalformulations to a patient, wherein the formulation is suitable forsubcutaneous administration. In further or additional embodiments, thepharmaceutical formulation is suitable for transcutaneousadministration.

In some embodiments, the pharmaceutical formulation comprises alipophilic long-acting selective beta-2 adrenergic receptor agonist, ora salt, optical isomer, racemate, solvate, or polymorph thereof and aglucocorticosteroid, or a salt, optical isomer, racemate, solvate, orpolymorph thereof. In certain embodiments, the glucorticosteroid isselected from fluticasone, mometasone, beclomethasone, triamcinolone,fluniolide, ciclesonide, or budesonide, or a salt, optical isomer,racemate, solvate, or polymorph thereof.

In other embodiments, the glucocorticosteroid is fluticasone or a salt,optical isomer, racemate, solvate, or polymorph thereof. In specificembodiments, the lipophilic long-acting selective beta-2 adrenergicreceptor agonist is salmeterol xinafoate and the glucocorticosteroid isfluticasone propionate. In yet another embodiment, theglucocorticosteroid is fluticasone furoate, or a salt, optical isomer,racemate, solvate, or polymorph thereof. In some embodiments, theglucocorticosteroid is fluticasone propionate (shown below as compound2), or its analogs, prodrugs, metabolites, and isomers thereof.

In one aspect, provided herein is an agent for reducing desensitizationof beta-adrenergic receptors and thereby increasing lipolysis,adipogenesis inhibition, and/or regional fat reduction duringbeta-adrenergic agonist exposure. In some embodiments, provided hereinis a pharmaceutical composition comprising at least one compound thatreduces desensitization of beta-adrenergic receptors, includingglucocorticosteroids, including for example fluticasone propionate orfluticasone furoate, whereby the compound is administered subcutaneouslyor transcutaneously, either alone (as single agent therapy), or incombination with at least a second active ingredient (as a combinationtherapy). Thus, in some embodiments, the pharmaceutical formulations(and corresponding methods of treatment provided herein) consistessentially of a glucocorticosteroid, for example, fluticasone,physiologically acceptable salts, optical isomers, racemates, solvates,polymorphs, or combinations thereof, wherein the formulation is suitablefor subcutaneous or transcutaneous administration. In further oradditional embodiments, the pharmaceutical formulation comprises aglucocorticosteroid, for example, fluticasone, physiologicallyacceptable salts, optical isomers, racemates, solvates, polymorphs, orcombinations thereof, and at least a second active ingredient, whereinthe formulation is suitable for subcutaneous or transcutaneousadministration.

In various embodiments, the lipophilic, long-acting selective beta-2adrenergic receptor agonists are administered separately or incombination with one or more compounds that reduce desensitization ofthe target tissue to the beta-adrenergic receptor agonist(s), forexample, glucocorticosteroids or ketotifen, or analogs thereof. The termdesensitization includes both short-term desensitization(tachyphylaxis), as well as long-term desensitization, as well asdesensitization over other time periods. Beta-2 adrenergic receptoragonists are also referred to herein as “beta-2 agonists” and “beta-2receptor agonists.” Unless otherwise specified, references to beta-2adrenergic receptor agonists also include their analogs, physiologicallyacceptable salts, optical isomers, racemates, solvates, and/orpolymorphs thereof. Some embodiments of the subcutaneous formulationcomprise from about 400:1 to about 1:400 lipophilic, long-actingselective beta-2 agonist to glucocorticosteroid. In further oradditional embodiments, the subcutaneous formulation comprise from about200:1 to about 1:200 lipophilic, long-acting selective beta-2 agonist toglucocorticosteroid.

In some embodiments, a formulation for use in the methods describedherein comprises a combination of the lipophilic, long-acting selectivebeta-2 agonists, such as salmeterol and physiologic salts, opticalisomers, racemates, solvates of polymorphs thereof and aglucocorticosteroid wherein the combination is suitable for subcutaneousadministration. Without wishing to be bound by theory, it is believedthat the combination of these compounds has an enhanced effect, by wayof a non-limiting example, in improving the appearance of regional fataccumulations and cellulite. Accordingly, provided herein aresynergistic pharmaceutical formulations that are suitable forsubcutaneous and/or transcutaneous administration to a patientcomprising a lipophilic, long-acting selective beta-2 agonists andcompounds that reduce desensitization of beta-adrenergic receptors. Insome embodiments, the combination of these two active ingredientsprovides a therapeutic effect when co-administered that is greater thanthe sum of the therapeutic effect when administered separately, i.e.when not co-administered.

Dosing of Active Ingredients

In some embodiments, long-term exposure of adipose tissue to adrenergicagents, particularly beta-adrenergic receptor agonists, results inreceptor desensitization through receptor phosphorylation andsequestration. These effects limit the ability of an adrenergicmodulating composition to treat adipose tissue and result intachyphylaxis, a condition in which the body experiences a rapidlydecreasing response to the agonist following administration of theinitial doses, to the desired lipolytic and anti-adipogenesis effect.Consequently, in certain situations with long-term exposure of adiposetissue to beta-adrenergic receptor agonists, the therapeutic effect withthe beta-adrenergic receptor agonists is short-lived.

Repeated administration of short-acting beta-2 agonists often result intachyphylaxis, as discussed above. However, salmeterol, exhibits partialbeta-2 receptor agonist activity in some systems that may reduce thedesensitization that occurs with continuous exposure of adipocytes tofull adrenergic receptor agonists. Compared with short-acting beta-2agonists, lipolysis also occurs for a longer time after administrationbecause lipophilic, long-acting selective beta-2 agonists have longerhalf-lives. The combination of longer half-lives and activities reducesthe required frequency and total dosage of administration of thepharmaceutical compositions provided herein. Consequently, in someembodiments, daily administration or more than once daily administrationof the composition is not required. In some embodiments, provided hereinare subcutaneously or transcutaneously administered adiposetissue-reducing lipophilic, long-acting selective beta-2 agonists whichexhibit greater selectivity for beta-2 receptors, permittingsubstantially similar therapeutic effects with less selective beta-2agonists at a lower dosage and/or less frequent dosage. Further the moreselective beta-2 activity can limit cardiac and other systemic sideeffects, which in the case of cardiac side effects, is often induced bybeta-1 receptor stimulation in the heart. In some embodiments, providedare subcutaneous or transcutaneous formulations of lipophilic,long-acting beta-2 agonists which provide selectivity for beta-2receptors while reducing the risk of producing cardiac or systemic sideeffects.

In some embodiments, beta-2 receptor activity or density increases inadipocytes within a regional fat deposit in response toglucocorticosteroid administration, particularly in the presence of abeta-adrenergic agonist. In some embodiments, increasing beta-2 receptoractivity and/or density potentiates the effect of long- and short-actingbeta-2 agonists. Thus, in some embodiments, the glucocorticosteroidsensitizes adipose tissue in a regional fat deposit to the effects ofbeta-2 receptor stimulation, e.g., lipolysis, inhibition ofadipogenesis, and/or apoptosis, and/or increases the ratio of beta-2adrenergic receptors to alpha-2 adrenergic receptors, thereby shiftingthe balance of the adipose tissue from fat accumulation to fat loss andresulting in reduction of the regional fat deposit. In some embodiments,beta-2 receptor number is increased or maintained especially with aglucocorticosteroid.

Provided herein are pharmaceutical formulations that are suitable forsubcutaneous or transcutaneous administration. In some embodiments, thepharmaceutical formulations provided herein are suitable forsubcutaneous injection, and provide for a volume of up to about 20 mL(including, e.g., about 0.1 mL, about 0.3 mL, about 0.5 mL, about 0.7mL, about 1.0 mL, about 1.1 mL, about 1.5 mL, about 2 mL, about 2.5 mL,about 3 mL, about 3.5 mL, about 4 mL, about 5 mL, about 6 mL, about 7mL, about 8 mL, about 9 mL, or any other volume from about 0.1 mL toabout 20 mL) of an excipient compatible with subcutaneousadministration. In some embodiments, the excipient concentration is keptbelow 1% (e.g., about 0.05%, about 0.2%, about 0.3%, about 0.4%, about0.5%, about 0.6%, about 0.8%, or any other concentration from about0.05% to less than about 1%.

Periodic Dosing Schedule

Another aspect of the formulations and methods of treatment providedherein is a periodic dosing schedule. It has been determined that onceper week dosing of a lipophilic long-acting selective beta-2 adrenergicreceptor agonist, e.g., salmeterol xinafoate, is more efficacious thantwice per week dosing. For example, it has been shown in a humanclinical trial that administering to a patient all of the activeingredient, including any sub-doses, in a single session dose thatoccurs once per week is more efficacious than administering to a patientthe active ingredient in two session doses per week. See, e.g., FIGS. 3Band 7B.

A periodic dose is the frequency at which a single session dose isadministered to a patient during a period. For example, in someembodiments, the periodic dose is once per week, and hence in thesesituations a patient will receive a single session dose once per week.In further or additional embodiments, the periodic dose is 2-7 times perweek (including any interval between 2 and 7), 3-6 times per week(including any interval between 3 and 6), or 4-5 days per week. In someembodiments, the periodic dose is 1-4 times per month (including anyinterval between 1 and 4), 2-3 times per month, or once or twice permonth. In some embodiments, the periodic dose is 1-52 times per year(including any interval between 1 and 52).

Because the single session doses provided herein are based on once perweek dosing, in situations where the periodic dose is different thanonce per week, in certain situations the single session dose amountadministered to the patient is normalized to account for thisdifference. For example, in some situations where the periodic dose istwice per week, the patient will receive the single session doses in twoseparate halves (that are about equal or unequal) during the weekcompared to what is provided herein. Similarly, in some situations wherethe periodic dose is seven times per week, the amount of activeingredient administered to the patient for each single session dosecompared to what is provided herein is divided by seven. As anotherexample, in certain situations where the periodic dose is once permonth, the patient will receive a single session dose per month at fourtimes the amount that is provided herein.

Single Session Dose

An additional aspect of the formulations and methods of treatmentprovided herein is a single session dose. A single session dose is thetotal amount of active ingredient administered to a patient during asingle visit for treatment by a healthcare professional or, insituations of self-administration, it is the amount of active ingredientadministered to the patient by self-administration in a single session.The single session doses provided herein are based on a once per weekperiodic dose, and can be adjusted for a different periodic dose thanonce per week as provided herein. As discussed herein, in someembodiments a single session dose includes 20 or more sub-doses.

It has been determined that of the patients tested in a clinical trial,the greatest therapeutic effectiveness (e.g., as indicated by reductionin adipose tissue and/or reduction in waist or abdomen circumference)was achieved in patients that received the least amount of thelipophilic long-acting beta-2 adrenergic receptor agonistco-administered with a glucocorticosteroid. See FIG. 4. As shown in FIG.4, of the cohorts tested, it has been determined that the greatesttherapeutic efficacy was achieved in patients administered the lowestsingle session dose of 0.5 μg of salmeterol xinafoate once per week.

Salmeterol

Accordingly, in one aspect, including certain methods of treatmentcomprising administration of the pharmaceutical formulations describedherein, provided are pharmaceutical formulations wherein a lipophiliclong-acting selective beta-2 adrenergic receptor agonist, either alone(as single agent therapy) or in combination with one or more additionalactive ingredients, including glucocorticosteroids (as a combinationtherapy), is provided in a single session dose that is less than about20 μg of the lipophilic long-acting selective beta-2 adrenergic receptoragonist, or a salt, optical isomer, racemate, solvate, or polymorphthereof. In some embodiments, the lipophilic, long-acting selectivebeta-2 agonist is salmeterol, or a salt, optical isomer, racemate,solvate, or polymorph thereof. In further embodiments, the lipophilic,long-acting selective beta-2 agonist is salmeterol xinafoate. In furtheror additional embodiments, the salmeterol xinafoate is administered in aperiodic dose that is once per week, and is administered to a patient insingle session dose as provided for herein.

In some embodiments, the single session dose of salmeterol xinafoate isadministered once per week in an amount between about 20 μg and about 5ng, between about 20 μg and about 25 ng, between about 20 μg and about50 ng, between about 20 μg and about 75 ng, between about 20 μg andabout 100 ng, between about 20 μg and about 125 ng, between about 20 μgand about 150 ng, between about between about 20 μg and about 175 ng,between about 20 μg and about 200 ng, between 20 μg and about 225 ng,between about 20 μg and about 250 ng, between about 20 μg and about 275ng, between about 20 μg and about 300 ng, between about 20 μg and about325 ng, between about 19 μg and about 350 ng, between about 19 μg andabout 375 ng, between about 18 μg and about 400 ng, between about 18 μgand about 425 ng, between about 18 μg and about 450 ng, between about 18μg and about 475 ng, between about 18 μg and about 500 ng, between about17 μg and about 525 ng, between about 17 μg and about 550 ng, betweenabout 17 μg and about 575 ng, between about 17 μg and about 600 ng,between about 17 μg and about 625 ng, between about 17 μg and about 650ng, between about 16 μg and about 675 ng, between about 15 μg and about700 ng, between about 14 μg and about 725 ng, between about 13 μg andabout 750 ng, between about 13 μg and about 775 ng, between about 13 μgand about 800 ng, between about 12 μg and about 825 ng, between about 11μg and about 850 ng, between about 10 μg and about 875 ng, between about9 μg and about 900 ng, between about 8 μg and about 920 ng, betweenabout 7 μg and about 940 ng, between about 6 μg and about 950 ng,between about 5 μg and about 960 ng, between about 4 μg and about 980ng, or between about 3 μg and about 1 g.

In still further embodiments, provided are pharmaceutical formulationsthat are formulated to provide a daily dose of a lipophilic, long-actingselective beta-2 agonist. In some embodiments, a lipophilic, long-actingselective beta-2 agonist to be administered is salmeterol and an adiposetissue-reducing amount of salmeterol to be administered is about 0.001μg/day to about 1000 μg/day, e.g., about 0.1 μg/day to about 100 μg/day,about 1 μg/day to about 100 μg/day, about 10 μg/day to about 100 μg/day,about 50 μg/day to about 100 μg/day, or any other dose of salmeterolfrom about 0.001 μg/day to about 1000 μg/day.

Fluticasone

Also provided herein, in further or additional embodiments, includingcertain methods of treatment comprising administration of thepharmaceutical formulations described herein, are pharmaceuticalformulations wherein a glucocorticosteroid, either alone (as singleagent therapy) or in combination with one or more additional activeingredients, for example a lipophilic, long-acting selective beta-2agonist (as a combination therapy), is provided for in a single sessiondose that is less than about 25 μg of the glucocorticosteroid, or asalt, optical isomer, racemate, solvate, or polymorph thereof. In someembodiments, the glucocorticosteroid is fluticasone, or a salt, opticalisomer, racemate, solvate, or polymorph thereof. In further embodiments,the glucocorticosteroid is fluticasone propionate or fluticasonefuroate.

In certain embodiments, provided are pharmaceutical formulations thatare formulated to provide a single session dose of fluticasonepropionate between about 25 μg and about 5 ng. In some embodiments, thepharmaceutical formulations provided herein comprise a single sessiondose that is an amount of fluticasone that is equal to or less thanabout 25 μg (including, e.g., about 5 ng, about 50 ng, about 500 ng,about 1 μg, about 10 μg, about 25 μg, or any other amount between about25 μg and about 5 ng).

In still further embodiments, provided are pharmaceutical formulationsthat are formulated to provide a daily dose of a glucocorticosteroid. Insome embodiments, a glucocorticosteroid to be administered isfluticasone and an adipose tissue-reducing amount of fluticasone to beadministered is about 0.001 μg/day to about 1000 μg/day, e.g., about 0.1μg/day to about 100 μg/day, about 1 μg/day to about 100 μg/day, about 10μg/day to about 100 μg/day, about 50 μg/day to about 100 μg/day, or anyother dose of fluticasone from about 0.001 μg/day to about 1000 μg/day.

In some embodiments, the glucocorticosteroid to be subcutaneously ortranscutaneously administered is fluticasone propionate and thetherapeutically effective amount of fluticasone propionate is from about0.05 μg/day to about 500 μg/day, e.g., about 0.05 μg/day to about 400μg/day, about 0.1 μg/day to about 300 μg/day, about 0.05 μg/day to about200 μg/day, about 1 μg/day to about 100 μg/day, about 2 μg/day to about50 μg/day, about 3 μg/day to about 20 μg/day, about 4 μg/day to about 10μg/day, about 5 μg/day to about 7 μg/day, about 1 μg/day to about 10μg/day, or about 2 μg/day to about 5 μg/day of fluticasone propionate,or any other dose of fluticasone propionate from about 0.05 μg/day toabout 500 μg/day. Other embodiments comprise the use of fluticasone or asalt, optical isomer, racemate, solvate or polymorph thereof, such as,by way of example only, the furoate salt of fluticasone. Still otherembodiments comprise the fluticasone furoate subcutaneously ortranscutaneously administered from about 0.05 μg/day to about 500μg/day, e.g., about 0.05 μg/day to about 400 μg/day, about 0.1 μg/day toabout 300 μg/day, about 0.05 μg/day to about 200 μg/day, about 1 μg/dayto about 100 μg/day, about 2 μg/day to about 50 μg/day, about 3 μg/dayto about 20 μg/day, about 4 μg/day to about 10 μg/day, about 5 μg/day toabout 7 μg/day, about 1 μg/day to about 10 μg/day, or about 2 μg/day toabout 5 μg/day of fluticasone furoate, or any other dose of fluticasonefuroate from about 0.05 μg/day to about 500 μg/day.

Salmeterol and Fluticasone

In an embodiment, provided herein is a subcutaneous injectableformulation that comprises a single session dose of salmeterol xinafoatebetween about 20 μg and about 5 ng, as described herein, including anyother amount of salmeterol xinafoate between about 20 μg and about 5 ngand between about 25 μg and about 50 ng of fluticasone propionate asdescribed herein, including any other amount of fluticasone propionatebetween about 25 μg and about 50 ng.

In still further embodiments, provided are pharmaceutical formulationsthat are formulated to provide a daily dose of a lipophilic, long-actingselective beta-2 agonist and glucocorticosteroid. In some embodiments, alipophilic, long-acting selective beta-2 agonist to be administered issalmeterol and an adipose tissue-reducing amount of salmeterol to beadministered is about 0.001 μg/day to about 1000 μg/day (including anydose of salmeterol from about 0.001 μg/day to about 1000 μg/day) and anadipose tissue-reducing amount of fluticasone to be administered isabout 0.001 μg/day to about 1000 μg/day (including any dose offluticasone from about 0.001 μg/day to about 1000 μg/day).

Sub-Dosing

In certain situations, the single session dose is administered to thepatient in sub-doses (e.g., by subcutaneous injection, transcutaneousapplication, or otherwise). Accordingly, in another aspect, includingcertain methods of treatment comprising administration of thepharmaceutical formulations described herein, provided arepharmaceutical formulations wherein a lipophilic long-acting selectivebeta-2 adrenergic receptor agonist, either alone (as single agenttherapy) or in combination with one or more additional activeingredients (as a combination therapy), is provided in at least twosub-doses. In some embodiments, all of the sub-doses are provided to apatient in a single single session during a single week. In further oradditional embodiments, including certain methods of treatmentcomprising administration of the pharmaceutical formulations describedherein, provided are pharmaceutical formulations wherein aglucocorticosteroid, either alone (as single agent therapy) or incombination with one or more additional active ingredients (as acombination therapy), is provided in at least two sub-doses whereby allof the sub-doses are provided to a patient in a single session during asingle week. In still further embodiments, at least at least twosub-doses are provided to a patient.

In some embodiments, one or more sub-dose is provided to a patientwherein each sub-dose is a single injection of a fluid comprising alipophilic long-acting selective beta-2 adrenergic receptor agonistand/or glucocorticosteroid. For example, in some embodiments, providedherein are pharmaceutical formulations and methods of treatmentcomprising administration of a pharmaceutical formulation wherein alipophilic long-acting selective beta-2 adrenergic receptor agonistand/or glucocorticosteroid is provided to a patient in about a singlesub-dose, at least about two sub-doses, at least about three sub-doses,at least about four sub-doses, at least about five sub-doses, at leastabout six sub-doses, at least about seven sub-doses, at least abouteight sub-doses, at least about nine sub-doses, at least about 10sub-doses, at least about 11 sub-doses, at least about 12 sub-doses, atleast about 13 sub-doses, at least about 14 sub-doses, at least about 15sub-doses, at least about 16 sub-doses, at least about 17 sub-doses, atleast about 18 sub-doses, at least about 19 sub-doses, at least about 20sub-doses, at least about 21 sub-doses, at least about 22 sub-doses, atleast about 23 sub-doses, at least about 24 sub-doses, at least about 25sub-doses, at least about 26 sub-doses, at least about 27 sub-doses, atleast about 28 sub-doses, at least about 29 sub-doses, at least about 30sub-doses, at least about 31 sub-doses, at least about 32 sub-doses, atleast about 33 sub-doses, at least about 34 sub-doses, at least about 35sub-doses, or more than about 35 sub-doses.

In some embodiments, each sub-dose is administered to a patient in anequal amount. For example, in some situations where the single sessiondose is about 20 μg of salmeterol xinafoate that is delivered to thepatient in 22 sub-doses, each sub-dose contains about 1 μg of salmeterolxinafoate. In other situations, the single session dose is about 500 ngand is delivered to the patient in 22 sub-doses and each sub-dosecontains about 22 or 23 ng of salmeterol xinafoate. Still in furthersituations, a prescribing physician may administer, or the patient mayself-administer, sub-doses in amounts that are not equal but vary inamount with respect to each sub-dose that is administered.

In some embodiments, at least two sub-doses of salmeterol xinafoate orfluticasone propionate, as described herein, are administered to apatient in a single session dose via subcutaneous injection to theabdominal region of the patient. In some of these embodiments, eachsub-dose is applied to a patient about 2-6 cm away from a closest secondsub-dose. In further embodiments, each sub-dose is applied to a patientabout 4 cm away from a closest second sub-dose. See, e.g., Example 3C.

In some embodiments, a sub-dose is administered, for example bysubcutaneous or transcutaneous injection, to areas of non-visceral fatdeposits on a subject, including for example subcutaneous fat. In someembodiments for which the formulations described herein are usefulinclude, but are not limited to, the inside region of the knees, themiddle to upper area of the upper arm, including the tricep area, thesubmental area, including the area under the chin, for example thewattle (which is understood to refer to the fleshy fold of skin in thesubmental area of a patient), the abdomen, the hips, the inner thigh,the outer thigh, the buttocks, the lower back, upper back and the chest.

Salmeterol

In certain embodiments, provided are pharmaceutical formulations andmethods of treatment comprising the pharmaceutical formulations that areformulated to provide a sub-dose of salmeterol xinafoate between about20 μg and about 1 ng, between about 20 μg and about 2 ng, between about20 μg and about 3 ng, between about 20 μg and about 4 ng, between about20 μg and about 5 ng, between about 20 μg and about 6 ng, between about20 μg and about 7 ng, between about 15 μg and about 8 ng, between about10 μg and about 9 ng, between about 5 μg and about 10 ng, between about1 μg and about 12 ng, between about 900 ng and about 14 ng, betweenabout 800 ng and about 16 ng, between about 700 ng and about 18 ng,between about 600 ng and about 20 ng, between about 550 ng and about 22ng, between about 500 ng and about 24 ng, between about 450 ng and about26 ng, between about 400 ng and about 28 ng, between about 350 ng andabout 30 ng, between about 300 ng and about 32 ng, between about 250 ngand about 34 ng, between about 200 ng and about 36 ng, between about 150ng and about 38 ng, between about 125 ng and about 40 ng, between about100 ng and about 42 ng, between about 90 ng and about 44 ng, betweenabout 80 ng and about 46 ng, between about 75 ng and about 48 ng,between about 70 ng and about 50 ng, between about 69 ng and about 51ng, between about 68 ng and about 52 ng, between about 67 ng and about53 ng, between about 66 ng and about 54 ng, between about 65 ng andabout 55 ng, between about 64 ng and about 56 ng, between about 63 ngand about 57 ng, between about 62 ng and about 58 ng, between about 61ng and about 59 ng, or about 60 ng.

In some embodiments, provided herein are pharmaceutical formulations,and methods of treatment comprising administration of the pharmaceuticalformulations, that are formulated to provide a sub-dose of salmeterolxinafoate that is equal to or less than about 20 μg, equal to or lessthan about 19 μg, equal to or less than about 18 μg, equal to or lessthan about 17 μg, equal to or less than about 16 μg, equal to or lessthan about 15 μg, equal to or less than about 14 μg, equal to or lessthan about 13 μg, equal to or less than about 12 μg, equal to or lessthan about 11 μg, equal to or less than about 10 μg, equal to or lessthan about 9 μg, equal to or less than about 8 μg, equal to or less thanabout 7 μg, equal to or less than about 6 μg, equal to or less thanabout 5 μg, equal to or less than about 4 μg, equal to or less thanabout 3 μg, equal to or less than about 2 μg, equal to or less thanabout 1 μg, equal to or less than about 975 ng, equal to or less thanabout 950 ng, equal to or less than about 925 ng, equal to or less thanabout 900 ng, equal to or less than about 875 ng, equal to or less thanabout 850 ng, equal to or less than about 825 ng, equal to or less thanabout 800 ng, equal to or less than about 775 ng, equal to or less thanabout 750 ng, equal to or less than about 725 ng, equal to or less thanabout 700 ng, equal to or less than about 675 ng, equal to or less thanabout 650 ng, equal to or less than about 625 ng, equal to or less thanabout 600 ng, equal to or less than about 575 ng, equal to or less thanabout 550 ng, equal to or less than about 525 ng, equal to or less thanabout 500 ng, equal to or less than about 475 ng, equal to or less thanabout 450 ng, equal to or less than about 425 ng, equal to or less thanabout 400 ng, equal to or less than about 375 ng, equal to or less thanabout 350 ng, equal to or less than about 325 ng, equal to or less thanabout 300 ng, equal to or less than about 275 ng equal to or less thanabout 250 ng, equal to or less than about 225 ng, equal to or less thanabout 200 ng, equal to or less than about 175 ng, equal to or less thanabout 150 ng, equal to or less than about 125 ng, equal to or less thanabout 100 ng, equal to or less than about 75 ng, or equal to or lessthan about 50 ng, or equal to or less than about 25 ng, equal to or lessthan about 20 ng, equal to or less than about 15 ng, equal to or lessthan about 10 ng, equal to or less than about 5 ng, or equal to or lessthan about 1 ng.

Suitable tissue concentration of salmeterol via subcutaneousadministration for adipose tissue treatment include from about 1 pM toabout 100 μM, e.g., about 0.01 μM to about 50 μM, 0.5 μM to about 50 μM,about 2.0 μM to about 50 μM, about 5 μM to about 50 μM, about 10 μM toabout 50 μM, about 20 μM to about 75 μM, or any other tissueconcentration of salmeterol from about 0.1 nM to about 100 μM.

Fluticasone

Also provided herein, in further or additional embodiments, includingcertain methods of treatment comprising administration of thepharmaceutical formulations described herein, are pharmaceuticalformulations wherein a glucocorticosteroid, either alone (as singleagent therapy) or in combination with one or more additional activeingredients, for example a lipophilic, long-acting selective beta-2agonist (as a combination therapy), is formulated to be administered toa patient in a sub-dose that is between about 25 μg and about 5 ng. Insome embodiments, the glucocorticosteroid is fluticasone, or a salt,optical isomer, racemate, solvate, or polymorph thereof. In furtherembodiments, the glucocorticosteroid is fluticasone propionate orfluticasone furoate.

In certain embodiments, provided are pharmaceutical formulations thatare formulated to provide a sub-dose of fluticasone propionate that isequal to or less than about 25 μg (including, e.g., about 5 ng, about 50ng, about 500 ng, about 1 μg, about 10 μg, about 25 μg, or any otheramount between about 25 μg and about 5 ng).

Appropriate tissue concentrations of glucocorticosteroids used for thetherapeutic methods described herein range from about 0.001 μM to about5 μM, e.g., from about 1.0 μM to about 5 μM, from about 0.1 μM to about2 μM, from about 0.1 μM to about 1 mM, from about 0.01 μM to about 0.1μM or any other tissue concentration of the glucocorticosteroid fromabout 0.001 μM to about 5 μM.

Salmeterol and Fluticasone

In a specific embodiment, provided herein is a subcutaneous injectableformulation that comprises a sub-dose of salmeterol xinafoate betweenabout 20 μg and about 1 ng, as described herein, including any otheramount of salmeterol xinafoate between about 20 μg and about 1 ng andbetween about 25 μg and about 5 ng of fluticasone propionate asdescribed herein, including any other amount of fluticasone propionatebetween about 25 μg and about 5 ng.

In one embodiment, a subcutaneous injectable formulation, or atranscutaneous formulation, that is formulated to be administered to apatient as a single session dose or a sub-dose that comprises from about20 μg to about 1 ng of salmeterol xinafoate (e.g., about 1 ng, about 50ng, about 250 ng, about 500 ng, about 750 ng, about 1 μg, about 5 μg,about 10 μg, about 15 μg, or about 20 μg, or any other amount ofsalmeterol xinafoate from about 20 μg to about 1 ng) and from about 300μg to about 5 ng of fluticasone propionate (e.g., about 5 ng, about 500ng, about 1 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg,about 60 μg, about 70 □μg, about 80 μg, about 90 μg, about 100 μg, orany other amount of fluticasone propionate from about 300 μg to about 5ng) formulated in a volume of up to about 10 mL (e.g., about 0.1 mL,about 0.3 mL, about 0.5 mL, about 0.7 mL, about 1.0 mL, about 1.1 mL,about 1.5 mL, about 2 mL, about 2.5 mL, about 3 mL, about 3.5 mL, about4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, or anyother volume from about 0.1 mL to about 10 mL) of an excipientcompatible with subcutaneous administration. In some embodiments, theexcipient concentration is kept below 1% (e.g., about 0.05%, about 0.2%,about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.8%, or any otherconcentration from about 0.05% to less than about 1%.

Pharmacokinetic Parameters

In another aspect is a pharmaceutical formulation, including certainmethods of treatment comprising administration of the pharmaceuticalformulations described herein, comprising an adipose tissue-reducingamount of salmeterol or a salt, optical isomer, racemate, solvate, orpolymorph thereof and at least one subcutaneously acceptable inactiveingredient, wherein the formulation provides a mean plasma salmeterolC_(max) equal to or less than about 300 pg/mL when administeredsubcutaneously. In one embodiment, the formulation provides a meanplasma salmeterol C_(max) equal to or less than about 270 pg/mL. In oneembodiment, the formulation provides a mean plasma salmeterol C_(max)equal to or less than about 250, about 230, about 200, about 190, about180, about 170, about 160, about 150, about 140, about 130, about 120,about 110, about 100, about 90, about 80, about 70, about 60, about 50,about 40, about 30, about 20, about 10, about 3, about 1 pg/mL, or isundetectable using conventional methodology. For purposes of thisapplication, “undetectable using conventional methodology” or“undetectable using current methodology,” means that the concentrationis lower than the low limit of quantitation (LLOQ) using the LiquidChromatography/Mass Spectrometry/Mass Spectrometry (LC/MS/MS) method,which is understood in the art to be a type of tandem mass spectrometryfor determining C_(max) levels.

In one embodiment, the pharmaceutical formulation further comprises aglucocorticosteroid or a salt, optical isomer, racemate, solvate, orpolymorph thereof. In another embodiment, the glucocorticosteroid isfluticasone or a pharmaceutically acceptable salt, optical isomer,racemate, solvate, or polymorph thereof. In a further embodiment, theglucocorticosteroid is fluticasone propionate. In yet anotherembodiment, the glucocorticosteroid is fluticasone furoate. In oneembodiment, the formulation provides a mean plasma fluticasone C_(max)of about 1 to about 200 pg/mL. In a further embodiment, the formulationprovides a mean plasma fluticasone propionate C_(max) of about 1 toabout 200 pg/mL. In yet a further embodiment, the mean plasmafluticasone propionate C_(max) is about 100, about 90, about 80, about70, about 60, about 50, about 40, about 30, about 20, about 10, about 3pg/mL, about 1 pg/mL, or is undetectable using conventional methodology.In a further embodiment, the mean plasma fluticasone propionate C_(max)is about 50 pg/mL. In another embodiment, the mean plasma fluticasonepropionate C_(max) results from subcutaneous administration. In anotherembodiment, salmeterol and fluticasone propionate are co-administeredtogether in a formulation suitable for subcutaneous administration. In afurther embodiment, salmeterol and fluticasone propionate areadministered separately in formulations suitable for subcutaneousadministration.

Partitioning Into Adipose Tissue

In a further aspect is a pharmaceutical formulation comprising anadipose tissue-reducing amount of salmeterol or a salt, optical isomer,racemate, solvate, or polymorph thereof and at least one subcutaneouslyacceptable inactive ingredient, wherein the formulation provides asalmeterol plasma C_(max) ratio of subcutaneous to intravenousadministration of between about 0.01 to about 0.4 when administeredsubcutaneously (also known as the “salmeterol partition” ratio.) Forpurposes of this application, the ratio of plasma C_(max) of along-acting beta-2 adrenergic receptor agonist administeredsubcutaneously to the plasma C_(max) of the same long-acting beta-2adrenergic receptor agonist administered intravenously is known as the“partition” ratio. Thus, in one embodiment, the partition ratio ofsalmeterol is about 0.01 to about 0.4. In another embodiment, thesalmeterol partition ratio is about 0.05 to about 0.3. In anotherembodiment, the salmeterol partition ratio is from about 0.1 to about0.35. In a further embodiment, the salmeterol partition ratio is about0.1. In another embodiment, the salmeterol partition ratio is between0.05 to about 0.2. In a further embodiment, the salmeterol partitionratio is between about 0.1 to about 0.2. In yet another embodiment, thesalmeterol partition ratio is about 0.01, about 0.02, about 0.03, about0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about0.1, about 0.11, about 0.12, about 0.13, about 0.14, about 0.015, about0.16, about 0.17, about 0.18, about 0.19, about 0.2, about 0.21, about0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about0.40. By way of a non-limiting example only, salmeterol xinafoateformulated in 5% PEG-400 in 0.9% saline USP, at various concentrationswere administered to non-naive Gottingen minipigs via single intravenousinjection or subcutaneous injection (see Example 1.) The salmeterolpartition ratio was calculated as the average salmeterol plasma C_(max)of subcutaneous administration ((403+575)/2) divided by the averagesalmeterol plasma C_(max) of intravenous administration ((4950+4290)/2).Thus, in this non-limiting example, the salmeterol partition ratio wasdetermined to be 0.1. In a further embodiment, is a subcutaneousformulation comprising of salmeterol or a salmeterol-like compound and asubcutaneously acceptable excipient wherein the formulation provides apartition ratio of between about 0.01 and about 0.4. As used herein, asalmeterol-like compound is a compound having a partition ratio ofbetween about 0.01 and 0.4 and provides limited systemic exposure, andconsequently, a reduced or minimized risk of producing cardiovascularside effects. Additionally, salmeterol-like compounds also selectivelypartition into the adipose tissue due to their lipophilic nature. Inanother embodiment, is a pharmaceutical formulation providing asalmeterol partition ratio of between about 0.01 to about 0.2 andfurther comprising a glucocorticosteroid or a salt or solvate thereof.In yet another embodiment, the glucocorticosteroid is fluticasonepropionate. In one embodiment, the formulation provides a salmeterolpartition ratio of between about 0.01 to about 0.3 and a mean plasmafluticasone propionate C_(max) of about 1 to about 200 pg/mL. In yet afurther embodiment, the formulation provides a salmeterol partitionratio of between about 0.01, about 0.02, about 0.03, about 0.04, about0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about0.11, about 0.12, about 0.13, about 0.14, about 0.015, about 0.16, about0.17, about 0.18, about 0.19, about 0.2 and a mean plasma fluticasonepropionate C_(max) of about 190, about 180, about 170, about 160, about150, about 130, about 120, about 110, about 100, about 90, about 80,about 70, about 60, about 50, about 40, about 30, about 20, about 10,about 1 pg/mL. In another embodiment, the mean plasma fluticasonepropionate C_(max) results from subcutaneous administration. In afurther embodiment, salmeterol and fluticasone propionate areco-administered together in a formulation suitable for subcutaneousadministration such that the formulation provides a salmeterol plasmaC_(max) ratio of subcutaneous to intravenous administration of betweenabout 0.01 to about 0.2 and a mean plasma fluticasone propionate C_(max)of about 1 to about 200 pg/mL. In a further embodiment, salmeterol andfluticasone propionate are administered separately in formulationssuitable for subcutaneous administration such that the formulationsprovide a salmeterol plasma C_(max) ratio of subcutaneous to intravenousadministration of between about 0.01 to about 0.2 and a mean plasmafluticasone propionate C_(max) of about 1 to about 200 pg/mL. In otherembodiments, fluticasone furoate is used as a glucocorticosteroid suchthat the formulation provides a salmeterol plasma C_(max) ratio ofsubcutaneous to intravenous administration of between about 0.01 toabout 0.2 and a mean plasma fluticasone furoate C_(max) of about 1 toabout 200 pg/mL.

In further embodiments, the pharmaceutical formulations provided hereinthat provide a salmeterol plasma C_(max) ratio of subcutaneous tointravenous administration of between about 0.01 to about 0.4 whensubcutaneously administered to a patient further provide a reduction inthe circumference of the patient's waist or abdomen. In someembodiments, provided herein is a formulation that is formulated toprovide a single session dose of salmeterol xinafoate in an amount thatis about 5 ng to about 20 μg and that provides a salmeterol plasmaC_(max) ratio of subcutaneous to intravenous administration of betweenabout 0.01 to about 0.4 when administered subcutaneously. In further oradditional embodiments, provided herein is a pharmaceutical formulationcomprising a single session dose of fluticasone propionate in an amountthat is between about 1 μg and about 300 μg. In some embodiments,provided herein are pharmaceutical formulations comprising a weekly doseof salmeterol xinafoate that is between about 5 ng to about 150 μg. Infurther or additional embodiments, provided herein is a pharmaceuticalformulation comprising a weekly dose of fluticasone propionate in anamount that is between about 50 ng and about 100 μg. In still furtherembodiments, provided herein is a pharmaceutical formulation comprisinga sub-dose of salmeterol xinafoate in an amount that is between about 1ng to about 50 μg. In other embodiments, provided herein is apharmaceutical formulation comprising a sub-dose of fluticasonepropionate in an amount that is between about 5 ng to about 20 μg.

In a further aspect is a subcutaneous formulation consisting essentiallyof a long-acting beta-2 receptor agonist and a subcutaneously acceptableexcipient thereof, wherein the formulation provides a partition ratiolower than the partition ratio of a reference long-acting beta-2receptor agonist. In one embodiment, the subcutaneous formulationprovides a partition ratio of about four to six times lower than thepartition ratio of a reference long-acting beta-2 receptor agonist. Inone embodiment, the formulation described herein provides a partitionratio about five times lower than the partition ratio of a referencelong-acting beta-2 receptor agonist. In another embodiment, thereference long-acting beta-2 receptor agonist has low lipophilicity. Inyet another embodiment, the reference long-acting beta-2 receptoragonist is formoterol. By way a non-limiting example only, formoterolfumarate dehydrate and budesonide were formulated in about 2% PEG-400 insaline and administered via single intravenous injection or subcutaneousinjection to non-naive Gottingen minipigs (see Example 2). The partitionratio was calculated as described above. Thus, in this non-limitingexample, the salmeterol formulation has a partition ratio about fivetimes lower than the partition ratio of formoterol (0.1 compared to0.5). In another embodiment, the subcutaneous formulation furthercomprises a glucocorticosteroid or a salt or solvate thereof. In yetanother embodiment, the glucocorticosteroid is fluticasone propionate.In a further embodiments, the glucocorticosteroid is fluticasonefuroate. In yet a further embodiment, the salmeterol formulationprovides a partition ratio about five times lower than a referencelong-acting beta-2 receptor agonist, wherein the reference long-actingbeta-2 adrenergic receptor agonist is formoterol, and a mean plasmafluticasone propionate C_(max) of about 1 to about 200 pg/mL. In anotherembodiment, the mean plasma fluticasone propionate C_(max) results fromsubcutaneous administration. In yet another embodiment, salmeterol andfluticasone propionate are co-administered together in a formulationsuitable for subcutaneous administration such that the formulationprovides a partition ratio of about five times lower than the partitionratio of formoterol and a mean plasma fluticasone propionate C_(max) ofabout 1 to about 200 pg/mL. In some embodiments, the fluticasonepropionate described in the formulations herein is substituted withfluticasone furoate such that, and by way of example only, theformulation provides a partition ratio about five times lower than thepartition ratio of formoterol and/or a mean plasma fluticasone furoateC_(max) of about 1 to about 200 pg/mL.

Methods of Reducing Adipose Tissue

In another aspect, provided herein are methods for reducing adiposetissue in a patient comprising administering to the patient apharmaceutical formulation comprising a lipophilic long-acting selectivebeta-2 adrenergic receptor agonist, either alone (as single agenttherapy) or in combination with one or more additional activeingredients, for example a glucocorticosteroid (as a combinationtherapy) and at least one subcutaneously acceptable inactive ingredient.In one aspect is a method for reducing adipose tissue in a subjectcomprising subcutaneously administering to the subject a pharmaceuticalformulation comprising an active agent consisting essentially of anadipose tissue-reducing amount of a lipophilic long-acting selectivebeta-2 adrenergic receptor agonist or a salt, optical isomer, racemate,solvate, or polymorph thereof and at least one subcutaneously acceptableinactive ingredient wherein adipose tissue in the subject is reduced. Inone embodiment, the lipophilic long-acting selective beta-2 adrenergicreceptor agonist is salmeterol. In yet another embodiment, the salt ofthe lipophilic long-acting selective beta-2 adrenergic agonist is axinafoate salt. In a further embodiment, provided is a method forreducing adipose tissue in a subject wherein the pharmaceuticalformulation further comprises a glucocorticosteroid or a salt or solvatethereof. In another embodiment, the glucocorticosteroid is fluticasoneor a salt or solvate thereof. In another embodiment, the salt is apropionate or furoate salt. In further embodiments, the salt is afuroate salt. In yet a further embodiment, the glucocorticosteroid isfluticasone propionate. In another embodiment, is a method for reducingadipose tissue in a subject wherein the pharmaceutical formulationprovides a mean plasma fluticasone propionate C_(max) between about 100pg/mL and a level that is undetectable using current methodology. In afurther embodiment, the subcutaneous administration results in a reducedor minimized risk of producing cardiovascular side effects.

In further embodiments, the pharmaceutical formulations provided hereinthat reduce adipose tissue when subcutaneously administered to a patientfurther provides a reduction in the circumference of the patient's waistor abdomen. In specific embodiments, the patient experiences a change inbody weight that is less than about 5%, less than about 3%, less thanabout 2%, less than about 1%, or less than about 0.5% during a treatmentperiod. In some embodiments, provided herein is a formulation that isformulated to provide a single session dose of salmeterol xinafoate inan amount that is about 5 ng to about 20 μg to a subject wherein adiposetissue in the subject is reduced. In further or additional embodiments,provided herein is a pharmaceutical formulation comprising a singlesession dose of fluticasone propionate in an amount that is betweenabout 1 μg and about 300 μg. In some embodiments, provided herein arepharmaceutical formulations comprising a weekly dose of salmeterolxinafoate that is between about 5 ng to about 150 μg. In further oradditional embodiments, provided herein is a pharmaceutical formulationcomprising a weekly dose of fluticasone propionate in an amount that isbetween about 50 ng and about 100 μg. In still further embodiments,provided herein is a pharmaceutical formulation comprising a sub-dose ofsalmeterol xinafoate in an amount that is between about 1 ng to about 50μg. In other embodiments, provided herein is a pharmaceuticalformulation comprising a sub-dose of fluticasone propionate in an amountthat is between about 5 ng to about 20 μg.

In yet a further embodiment, the subcutaneous administration results ina reduced or minimized risk of producing an increase in heart rate or adecrease in blood pressure or a combination thereof. In anotherembodiment, the subcutaneous administration provides a minimized risk ofproducing anaphylaxis related effects, such as, flushing, reddening,rapid heart rate, chest tightness, difficulty breathing, faintness,heart palpitations, hives, atrophy, pigmentation, nodularity, necrosis,irregular or abnormal heart rate, paroxysmal bronchoconstriction, andhypersensitivity reaction such as angioedema and urticaria. By way of anon-limiting example only, a subcutaneous formulation comprisingsalmeterol was administered to Gottingen minipigs. The salmeterolpartition ratio is determined as described above. Thus, in thisnon-limiting example, the subcutaneous formulation administered tominipigs provides a salmeterol partition ratio of 0.1 and a reduced orminimized risk of producing cardiovascular side effects. It should benoted that the reduced or minimized risk described herein (due tolimited systemic exposure) refers to a generalized population and mayvary depending on the individual subject or patient. Subcutaneousformulations consisting essentially of salmeterol and fluticasoneprovide therapeutic effect to a regional fat deposit and a reduced orminimized risk of producing the side effects associated with the use ofother long-acting beta-2 agonists or long-acting beta-2 agonistsadministered by other methods. Such side effects include paradoxicalbronchospasm, high blood pressure, abnormal heart rhythm, abnormally lowblood pressure, an increase in asthma related conditions, bronchospasm,inflammation of the lining of the stomach and intestines, involuntaryquivering, fast heartbeat, chest pain, and giant hives.

Methods of Treating Regional Fat Accumulation

In yet another aspect, provided herein are methods for treating regionalfat accumulation in a patient comprising administering to the patient apharmaceutical formulation comprising a lipophilic long-acting selectivebeta-2 adrenergic receptor agonist, either alone (as single agenttherapy) or in combination with one or more additional activeingredients, for example a glucocorticosteroid (as a combinationtherapy) and at least one subcutaneously acceptable inactive ingredient.In further embodiments, the method for treating regional fataccumulation in a subject comprises subcutaneously administering to aregional fat accumulation area a pharmaceutical formulation comprising aregional fat accumulation reducing amount of salmeterol or a salt,optical isomer, racemate, solvate, or polymorph thereof and at least onesubcutaneously acceptable inactive ingredient. In further or additionalembodiments, the formulation provides a mean plasma salmeterol C_(max)equal that is in the range of about 300 pg/mL to levels that areundetectable using conventional methodology, wherein the regional fataccumulation in the subject is reduced. In one embodiment, salmeterolselectively partitions into adipose tissue of the regional fataccumulation relative to plasma.

In one embodiment, provided is a method for treating regional fataccumulation in a subject comprising administration of thepharmaceutical formulations provided herein that when subcutaneouslyadministered to a patient, provides a reduction in the circumference ofthe patient's waist or abdomen. In further or additional embodiments,the patient experiences a change in body weight that is less than about5%, less than about 3%, less than about 2%, less than about 1%, or lessthan about 0.5% during a treatment period. In yet another embodiment,provided is a method for treating regional fat accumulation in a subjectcomprising subcutaneously administering to a regional fat accumulationarea, a pharmaceutical formulation comprising a regional fataccumulation reducing amount of salmeterol or a salt, optical isomer,solvate, or polymorph thereof and at least one subcutaneously acceptableinactive ingredient, wherein the formulation provides a mean plasmasalmeterol C_(max) equal that is in the range of about 300 pg/mL tolevels that are undetectable using conventional methodology. In furtheror additional embodiments, the pharmaceutical formulation furthercomprises a glucocorticosteroid or a salt, optical isomer, racemate,solvate, or polymorph thereof wherein the regional fat accumulation inthe subject is reduced. In one embodiment, the glucocorticosteroid isfluticasone or a salt or solvate thereof. In another embodiment, theglucocorticosteroid is fluticasone propionate. In a further embodimentthe glucocorticosteroid is fluticasone furoate.

In further embodiments, the method for treating regional fataccumulation in a subject comprises subcutaneously administering to aregional fat accumulation area a pharmaceutical formulation comprising aregional fat accumulation reducing amount of salmeterol or a salt,optical isomer, racemate, solvate, or polymorph thereof and at least onesubcutaneously acceptable inactive ingredient wherein the regional fataccumulation reducing amount is a single session dose of salmeterolxinafoate in an amount that is about 5 ng to about 20 μg. In further oradditional embodiments, provided herein is a pharmaceutical formulationcomprising a single session dose of fluticasone propionate in an amountthat is between about 1 μg and about 300 μg. In some embodiments,provided herein are pharmaceutical formulations comprising a weekly doseof salmeterol xinafoate that is between about 5 ng to about 150 μg. Infurther or additional embodiments, provided herein is a pharmaceuticalformulation comprising a weekly dose of fluticasone propionate in anamount that is between about 50 ng and about 100 μg. In still furtherembodiments, provided herein is a pharmaceutical formulation comprisinga sub-dose of salmeterol xinafoate in an amount that is between about 1ng to about 50 μg. In other embodiments, provided herein is apharmaceutical formulation comprising a sub-dose of fluticasonepropionate in an amount that is between about 5 ng to about 20 μg.

In yet another embodiment, provided is a method for treating regionalfat accumulation in a subject comprising subcutaneously administering toa regional fat accumulation area a pharmaceutical formulation comprisinga regional fat accumulation reducing amount of salmeterol or a salt,optical isomer, racemate, solvate, or polymorph thereof and furthercomprising fluticasone propionate, wherein the formulation provides amean plasma salmeterol C_(max) equal to or less than about 300 pg/mL(including C_(max) levels that are undetectable using conventionalmethodology) and a mean plasma fluticasone propionate C_(max) of about 1to about 100 pg/mL (including C_(max) levels that are undetectable usingconventional methodology). In one embodiment, provided is a method fortreating regional fat accumulation comprising subcutaneouslyadministering to a regional fat accumulation area a pharmaceuticalformulation comprising a regional fat accumulation reducing amount ofsalmeterol or a salt, optical isomer, racemate, solvate, or polymorphthereof and fluticasone propionate or a salt or solvate thereof whereinthe formulation provides a mean plasma fluticasone propionate C_(max)equal to or less than about 100, about 90, about 80, about 70, about 60,about 50, about 40, about 30, about 20, about 10, about 1 pg/mL, or isundetectable using conventional methodology. In other embodiments,provided are methods for treating regional fat accumulation comprisingsubcutaneously administering to a regional fat accumulation area apharmaceutical formulation comprising a regional fat accumulationreducing amount of salmeterol or a salt, optical isomer, racemate,solvate, or polymorph thereof and fluticasone or a salt or solvatethereof wherein the formulation provides a mean plasma fluticasoneC_(max) equal to or less than about 100, about 90, about 80, about 70,about 60, about 50, about 40, about 30, about 20, about 10, about 1pg/mL, or is undetectable using conventional methodology. In furtherembodiments, the fluticasone salt is a furoate salt. In furtherembodiments, the pharmaceutical formulations provided herein, whensubcutaneously administered to a patient, provide a reduction in thecircumference of the patient's waist. In specific embodiments, thepatient experiences a change in body weight that is less than about 5%,less than about 3%, less than about 2%, less than about 1%, or less thanabout 0.5% during a treatment period.

Also described herein is a method for treating regional fat accumulationcomprising subcutaneously administering to the subject a pharmaceuticalformulation comprising an active agent consisting essentially of aregional fat accumulation reducing amount of a long-acting selectivebeta-2 adrenergic receptor agonist or a salt, optical isomer, racemate,solvate, or polymorph thereof and at least one subcutaneously acceptableinactive ingredient. In one embodiment, the lipophilic long-actingselective beta-2 adrenergic receptor agonist is salmeterol. In anotherembodiment, the salt of the lipophilic long-acting selective beta-2adrenergic agonist is a xinafoate salt. In a further embodiment, thesubcutaneous administration reduces or minimizes the risk of producingcardiovascular side effects (by minimizing systemic exposure). In yet afurther embodiment, the subcutaneous administration reduces or minimizesthe risk of producing an increase in heart rate or a decrease in bloodpressure or a combination thereof. In another embodiment, the risks thatare reduced or minimized as a result of subcutaneous administrationinclude effects, such as, flushing, reddening, rapid heart rate, chesttightness, difficulty breathing, faintness, heart palpitations, hives,irregular or abnormal heart rate, paroxysmal bronchoconstriction, andhypersensitivity reaction such as angioedema and urticaria.

In yet another embodiment, provided is a method for treating regionalfat accumulation comprising subcutaneously administering to the subjecta pharmaceutical formulation comprising an adipose tissue-reducingamount of a lipophilic long-acting selective beta-2 adrenergic receptoragonist or a salt, optical isomer, racemate, solvate, or polymorphthereof and at least one subcutaneously acceptable inactive ingredientwherein the formulation provides a mean plasma lipophilic, long-actingselective beta-2 adrenergic receptor agonist C_(max) equal to or lessthan about 300 pg/mL (including C_(max) levels that are undetectableusing conventional methodology). In another embodiment, the lipophilic,long-acting selective beta-2 adrenergic receptor agonist is notformoterol. In one embodiment, the lipophilic long-acting selectivebeta-2 adrenergic receptor agonist is salmeterol. In yet anotherembodiment, the salt of salmeterol is a xinafoate salt. In yet anotherembodiment, is a method for treating regional fat accumulationcomprising subcutaneously administering to the subject a pharmaceuticalformulation comprising an active agent consisting essentially of aregional fat accumulation reducing amount of a lipophilic long-actingselective beta-2 adrenergic receptor agonist or a salt, optical isomer,racemate, solvate, or polymorph thereof and at least one subcutaneouslyacceptable inactive ingredient wherein the formulation provides a meanplasma lipophilic, long-acting selective beta-2 adrenergic receptoragonist C_(max) equal to or less than about 300 pg/mL (including C_(max)levels that are undetectable using conventional methodology) and whereinthe pharmaceutical formulation further comprises a glucocorticosteroidor a salt or solvate thereof. In one embodiment, the glucocorticosteroidis fluticasone propionate. In yet another embodiment, provided is amethod for treating regional fat accumulation comprising subcutaneouslyadministering to the subject a pharmaceutical formulation comprising aregional fat accumulation reducing amount of a lipophilic long-actingselective beta-2 adrenergic receptor agonist or a salt, optical isomer,racemate, solvate, or polymorph thereof and at least onepharmaceutically acceptable inactive ingredient and further comprisingfluticasone propionate, wherein the pharmaceutical formulation providesa plasma lipophilic, long-acting selective beta-2 adrenergic receptoragonist C_(max) equal to or less than about 300 pg/mL (including C_(max)levels that are undetectable using conventional methodology) and a meanplasma fluticasone propionate C_(max) equal to or less than about 100pg/mL (including C_(max) levels that are undetectable using conventionalmethodology). In one embodiment, is a method for treating regional fataccumulation comprising subcutaneously administering to the subject apharmaceutical formulation comprising a lipophilic, long-actingselective beta-2 adrenergic receptor agonist or a salt, optical isomer,racemate, solvate, or polymorph thereof and fluticasone propionatewherein the formulation provides a mean plasma fluticasone propionateC_(max) equal to or less than about 100, about 90, about 80, about 70,about 60, about 50, about 40, about 30, about 20, about 10, about 1pg/mL, or is undetectable using conventional methodology. In oneembodiment, the mean plasma fluticasone propionate C_(max) results fromsystemic administration. In another embodiment, the mean plasmafluticasone propionate C_(max) results from subcutaneous administration.In other embodiments, the formulation comprises a fluticasone salt, suchas by way of example only, fluticasone furoate, such that the meanplasma fluticasone furoate C_(max) results from systemic administration.In another embodiment, the mean plasma fluticasone furoate C_(max)results from subcutaneous administration. In yet other embodiments, themean plasma fluticasone results from fluticasone furoate.

Methods of Inducing Lipolysis in Adipose Tissue

As discussed herein, lipolysis and/or inhibition of adipogenesis arestimulated by the beta-1, 2, or 3 receptor subtypes. Thus, agonists toone, two and/or all three receptors are capable of stimulating lipolysisand/or inhibition of adipogenesis. In humans, beta-2 receptor activityis believed to be more important for stimulating lipolysis, particularlyin the presence of a glucocorticosteroid. Lipolytic activity andadipocyte proliferation inhibition are believed to be mediated throughmodulation of adrenergic receptors in adipose tissue and/or onadipocytes.

In another aspect, provided herein is a method for inducing lipolysis inadipose tissue comprising subcutaneously administering a pharmaceuticalformulation suitable for subcutaneous injection comprising: (a) alipophilic long-acting selective beta-2 adrenergic receptor agonist orglucocorticosteroid, or a salt, optical isomer, racemate, solvate, orpolymorph thereof; and (b) at least one subcutaneously acceptableinactive ingredient. In one aspect is a method for inducing lipolysis ina subject comprising subcutaneously administering to the subject apharmaceutical formulation comprising an active agent consistingessentially of an adipose tissue-reducing amount of a lipophiliclong-acting selective beta-2 adrenergic receptor agonist or a salt,optical isomer, racemate, solvate, or polymorph thereof and at least onesubcutaneously acceptable inactive ingredient that induced lipolysis ina patient, and reduces adipose tissue in the area treated. In oneembodiment, the lipophilic long-acting selective beta-2 adrenergicreceptor agonist is salmeterol. In yet another embodiment, the salt ofthe lipophilic long-acting selective beta-2 adrenergic agonist is axinafoate salt. In a further embodiment, provided is a method forinducing lipolysis in a subject wherein the pharmaceutical formulationfurther comprises a glucocorticosteroid or a salt or solvate thereof. Inanother embodiment, the glucocorticosteroid is fluticasone or a salt orsolvate thereof. In another embodiment, the salt is a propionate orfuroate salt. In further embodiments, the pharmaceutical formulationsprovided herein, when subcutaneously administered to a patient, providea reduction in the circumference of the patient's waist.

In further embodiments, the salt is a furoate salt. In yet a furtherembodiment, the glucocorticosteroid is fluticasone propionate. Inanother embodiment, is a method for reducing adipose tissue in a subjectwherein the pharmaceutical formulation provides a mean plasmafluticasone propionate C_(max) between about 100 pg/mL and a level thatis undetectable using current methodology. In a further embodiment, thesubcutaneous administration results in a reduced or minimized risk ofproducing cardiovascular side effects. In yet a further embodiment, thesubcutaneous administration results in a reduced or minimized risk ofproducing an increase in heart rate or a decrease in blood pressure or acombination thereof. In another embodiment, the subcutaneousadministration provides a minimized risk of producing effects, such as,flushing, reddening, rapid heart rate, chest tightness, difficultybreathing, faintness, heart palpitations, hives, irregular or abnormalheart rate, paroxysmal bronchoconstriction, and hypersensitivityreaction such as angioedema and urticaria. Other reduced or minimizedrisk side effects include paradoxical bronchospasm, high blood pressure,abnormal heart rhythm, abnormally low blood pressure, an increase inasthma related conditions, bronchospasm, inflammation of the lining ofthe stomach and intestines, involuntary quivering, fast heartbeat, chestpain, and giant hives.

In some embodiments, provided herein is a pharmaceutical formulationthat comprises salmeterol or a salt, optical isomer, racemate, solvate,or polymorph thereof and at least one subcutaneously acceptable inactiveingredient wherein the salmeterol is formulated in a single session dosethat is between about 5 ng to about 20 μg and when subcutaneouslyadministered to a patient, induces lipolysis of adipose tissue. Infurther or additional embodiments, provided herein is a pharmaceuticalformulation comprising a single session dose of fluticasone propionatein an amount that is between about 1 μg and about 300 μg. In someembodiments, provided herein are pharmaceutical formulations comprisinga weekly dose of salmeterol xinafoate that is between about 5 ng toabout 150 μg. In further or additional embodiments, provided herein is apharmaceutical formulation comprising a weekly dose of fluticasonepropionate in an amount that is between about 50 ng and about 100 μg. Instill further embodiments, provided herein is a pharmaceuticalformulation comprising a sub-dose of salmeterol xinafoate in an amountthat is between about 1 ng to about 50 μg. In other embodiments,provided herein is a pharmaceutical formulation comprising a sub-dose offluticasone propionate in an amount that is between about 5 ng to about20 μg.

In further or additional embodiments, the formulation provides a meanplasma lipophilic, long-acting selective beta-2 adrenergic receptoragonist C_(max) equal to or less than about 300 pg/mL (including C_(max)levels that are undetectable using conventional methodology). In oneembodiment, the lipophilic, long-acting selective beta-2 adrenergicreceptor agonist is not formoterol. In one embodiment, provided is amethod for inducing lipolysis in adipose tissue comprisingsubcutaneously administering to the subject a pharmaceutical formulationcomprising a lipophilic, long-acting selective beta-2 adrenergicreceptor agonist or a salt, optical isomer, racemate, solvate, orpolymorph thereof and fluticasone propionate wherein the formulationprovides a mean plasma fluticasone propionate C_(max) equal to or lessthan about 100, about 90, about 80, about 70, about 60, about 50, about40, about 30, about 20, about 10, about 1 pg/mL, or is undetectableusing conventional methodology. In one embodiment, the mean plasmafluticasone propionate C_(max) results from systemic administration. Inanother embodiment, the mean plasma fluticasone propionate C_(max)results from subcutaneous administration. In other embodiments, theformulation comprises a fluticasone salt, such as by way of exampleonly, fluticasone furoate, such that the mean plasma fluticasone furoateC_(max) results from systemic administration. In another embodiment, themean plasma fluticasone furoate C_(max) results from subcutaneousadministration. In yet other embodiments, the mean plasma fluticasoneresults from fluticasone furoate.

Methods of Reducing the Circumference of a Patient's Abdomen

It has been discovered that the subcutaneous administration of alipophilic long-acting selective beta-2 adrenergic receptor agonist,e.g., salmeterol xinafoate, to a patient provides for a reduction in thecircumference of the patient's waist or abdomen. In specificembodiments, the patient experiences a change in body weight that isless than about 5%, less than about 3%, less than about 2%, less thanabout 1%, or less than about 0.5% during a treatment period. Forexample, FIG. 8 illustrates that the mean body weight (in kg) did notshow any significant change when measured at baseline, 4 weeks fromstart of treatment (i.e., end of treatment), 1 week post-treatment, and4 weeks post-treatment, for all patients enrolled in the study describedin Example 3B. See FIG. 8.

Accordingly, in another aspect, provided herein is a method for reducingthe circumference of a patient's waist comprising subcutaneouslyadministering a pharmaceutical formulation suitable for subcutaneousinjection comprising: (a) a lipophilic long-acting selective beta-2adrenergic receptor agonist or glucocorticosteroid, or a salt, opticalisomer, racemate, solvate, or polymorph thereof; and (b) at least onesubcutaneously acceptable inactive ingredient wherein the patientexperiences a change in body weight that is less than about 5%, lessthan about 3%, less than about 2%, less than about 1%, or less thanabout 0.5% during a treatment period. In some embodiments, thecircumference of the patient's waist is reduced by at least about 1centimeter, at least about 1.5 centimeters, at least about 2centimeters, at least about 2.5 centimeters, at least about 3centimeters, at least about 3.5 centimeters, at least about 4centimeters, at least about 4.5 centimeters, or at least about 5centimeters. In some embodiments, the circumference of the patient'swaist is reduced by at least about two centimeters. In some embodiments,the circumference of the patient's waist is reduced by at least about2.5 centimeters. In further or additional embodiments, the reduction inthe patient's waist is evident at about 6 to 8 weeks from the first dayof treatment. In some embodiments, the lipophilic long-acting selectivebeta-2 adrenergic receptor agonist is salmeterol xinafoate.

In another embodiment, provided herein is a method for reducing thecircumference of a patient's waist or abdomen comprising thesubcutaneous administration of a glucocorticosteroid that is fluticasoneor a salt or solvate thereof. In another embodiment, the salt is apropionate or furoate salt. In further embodiments, the salt is afuroate salt. In yet a further embodiment, the glucocorticosteroid isfluticasone propionate. In another embodiment, is a method for reducingadipose tissue in a subject wherein the pharmaceutical formulationprovides a mean plasma fluticasone propionate C_(max) between about 100pg/mL and a level that is undetectable using current methodology. In afurther embodiment, the subcutaneous administration results in a reducedor minimized risk of producing cardiovascular side effects. In yet afurther embodiment, the subcutaneous administration results in a reducedor minimized risk of producing an increase in heart rate or a decreasein blood pressure or a combination thereof. In another embodiment, thesubcutaneous administration provides a minimized risk of producinganaphylaxis related effects, such as, flushing, reddening, rapid heartrate, chest tightness, difficulty breathing, faintness, heartpalpitations, hives, irregular or abnormal heart rate, paroxysmalbronchoconstriction, and hypersensitivity reaction such as angioedemaand urticaria. Other reduced or minimized risk side effects includeparadoxical bronchospasm, high blood pressure, abnormal heart rhythm,abnormally low blood pressure, an increase in asthma related conditions,bronchospasm, inflammation of the lining of the stomach and intestines,involuntary quivering, fast heartbeat, chest pain, and giant hives.

In some embodiments, provided herein is a pharmaceutical formulationthat comprises salmeterol or a salt, optical isomer, racemate, solvate,or polymorph thereof and at least one subcutaneously acceptable inactiveingredient wherein the salmeterol is formulated in a single session dosethat is between about 5 ng to about 20 μg. In specific embodiments, whensubcutaneously administered to a patient, the formulations providedherein provide a reduction in the circumference of the patient's waistand the patient experiences a change in body weight that is less thanabout 5%, less than about 3%, less than about 2%, less than about 1%, orless than about 0.5% during a treatment period. In further or additionalembodiments, provided herein is a pharmaceutical formulation comprisinga single session dose of fluticasone propionate in an amount that isbetween about 1 μg and about 300 μg. In some embodiments, providedherein are pharmaceutical formulations comprising a weekly dose ofsalmeterol xinafoate that is between about 5 ng to about 150 μg. Infurther or additional embodiments, provided herein is a pharmaceuticalformulation comprising a weekly dose of fluticasone propionate in anamount that is between about 50 ng and about 100 μg. In still furtherembodiments, provided herein is a pharmaceutical formulation comprisinga sub-dose of salmeterol xinafoate in an amount that is between about 1ng to about 50 μg. In other embodiments, provided herein is apharmaceutical formulation comprising a sub-dose of fluticasonepropionate in an amount that is between about 5 ng to about 20 μg.

In further or additional embodiments, provided herein is a method forreducing the circumference of a patient's waist wherein the formulationprovides a mean plasma lipophilic, long-acting selective beta-2adrenergic receptor agonist C_(max) equal to or less than about 300pg/mL (including C_(max) levels that are undetectable using conventionalmethodology). In another embodiment, the lipophilic, long-actingselective beta-2 adrenergic receptor agonist is not formoterol. In oneembodiment, provided is a method for inducing lipolysis in adiposetissue comprising subcutaneously administering to the subject apharmaceutical formulation comprising a lipophilic, long-actingselective beta-2 adrenergic receptor agonist or a salt, optical isomer,racemate, solvate, or polymorph thereof and fluticasone propionatewherein the formulation provides a mean plasma fluticasone propionateC_(max) equal to or less than about 100, about 90, about 80, about 70,about 60, about 50, about 40, about 30, about 20, about 10, about 1pg/mL, or is undetectable using conventional methodology. In oneembodiment, the mean plasma fluticasone propionate C_(max) results fromsystemic administration. In another embodiment, the mean plasmafluticasone propionate C_(max) results from subcutaneous administration.In other embodiments, the formulation comprises a fluticasone salt, suchas by way of example only, fluticasone furoate, such that the meanplasma fluticasone furoate C_(max) results from systemic administration.In another embodiment, the mean plasma fluticasone furoate C_(max)results from subcutaneous administration. In yet other embodiments, themean plasma fluticasone results from fluticasone furoate.

Pharmaceutically Acceptable Excipients

In a further embodiment, the at least one subcutaneously acceptableinactive ingredient is a co-solvent. In further or additionalembodiments, the co-solvent is selected from about 0.25 to about 40%polyethylene glycol. In further or additional embodiments, thepolyethylene glycol is about 0.8-about 1%. In a further embodiment, thepolyethylene glycol is PEG 400.

In yet a further embodiment, the at least one subcutaneously acceptableinactive ingredient is selected from about 0.01 to about 10%polysorbate. In some embodiments, the polysorbate is about 0.01-about 2%polysorbate. In still further embodiments, the polysorbate is about0.04%. In one embodiment, the polysorbate is polysorbate 80.

Excipients used in the formulations described herein include, but arenot limited to, suspending agents, surfactants, solubilizers such as,for example, PEG 400, stabilizers, diluents and the like, and should beselected on the basis of compatibility with the lipophilic long-actingbeta-2 adrenergic receptor agonist and/or in cases of a combinationformulation for subcutaneous administration, glucocorticosteroids. Insome embodiments, a polyalkylene glycol or a mixture of differentpolyalkylene glycols is used as a solubilizer. Polyalkylene glycols fromthe group of polypropylene glycols or polyethylene glycols areparticularly suitable in this connection because of the physiologicaltolerance. In this connection, the use of polyethylene glycols isutilized in some embodiments presented herein. In some embodiments,polyethylene glycols such as, for example, PEG 400, is contemplatedherein.

Additives increasing the bioavailability of a lipophilic long-actingbeta-2 adrenergic receptor agonist, such as, salmeterol are, in someembodiments, organic compounds, salts thereof, optical isomers orracemates thereof, emulsions or dispersions containing organic compoundsor salts thereof, e.g. dispersions of polar lipids, or any combination.Organic compounds useful in the subcutaneous formulation are e.g. aminoacids, peptides, proteins, and polysaccharides. Peptides includedipeptides, tripeptides, oligopeptides, such as collagen and gelatine.In some embodiments, the collagen and gelatine is hydrolyzed.Polysaccharides include e.g., chitosans, cyclodextrins, starch,hyaluronic acids, dextrans, cellulose, and any derivatives,combinations. In further embodiments, the starch is hydrolyzed. Theemulsions include oil-in-water emulsions with oil as the dispersed phaseand water-in-oil dispersions with oil as the continuous phase. In otherembodiments, the oil is of vegetable or of animal origin orsynthetically produced. In further embodiments, the polar liquids areone or more phospholipids or glycolipids or any combination thereof. Insome other embodiments, the additives increasing the bioavailability ofa lipophilic long-acting beta-2 adrenergic receptor agonist, such as,salmeterol are added to a stable solution or dispersion containing thelipophilic long-acting beta-2 adrenergic receptor agonist.

In further embodiments, before administration, one or more aqueoussolutions or dispersions are added, in any mixture or sequence, to thelipophilic long-acting beta-2 adrenergic receptor agonist, such as,salmeterol, which is a stable aqueous solution. In other embodiments,the formulation is a stable aqueous solution ready for administration.In some embodiments, it is a dispersion, e.g. a suspension, a liposomalformulation or an emulsion. In yet other embodiments, the formulationalso comprises a salt in order to give an isotonic solution, e.g., NaCl,KCl, and/or in further embodiments, it comprises one or more otherisotonicity establishing compounds.

In yet other embodiments, an amino acid is used to buffer the system. Insome embodiments, a suitable buffer is glycine, lysine, arginine,histidine or glycylglycine, in other embodiments, the buffer isglycylglycine.

In some other embodiments, a non-ionic surfactant is also present in theformulation. In some embodiments, the surfactant is chosen fromblock-copolymers, such as a poloxamer, e.g., poloxamer 188, or apolyoxyethylene sorbitan fatty acid ester, such aspolyoxyethylene-(20)-sorbitan monolaurate orpolyoxyethylene-(20)-sorbitan monooleate. Also disclosed herein areformulations using polyoxyethylene-(20)-sorbitan monolaurate (Tween 20).In one embodiment, the formulation described herein usedpolyoxyethylene-(20)-sorbitan monooleate (Tween 80). In otherembodiments, the non-ionic surfactant, is present in an amount above thecritical micelle concentration (CMC).

Also presented herein are mono- or disaccharides (e.g., sucrose),polysaccharides such as low molecular weight dextrins, or sugar alcohols(e.g., sorbitol, glycerol or mannitol) used in the subcutaneousformulations. In some embodiments, the formulation also comprisesantioxidants such as bisulfite, ascorbate glutathione, acetylcystein,tocopherol, methionin, EDTA, citric acid, butyl hydroxy toluene and/orbutyl hydroxy anisole. In other embodiments, complexing agents, such asEDTA and citric acid are also present in small concentrations forstabilizing the lipophilic long-acting beta-2 adrenergic receptoragonist, such as, salmeterol. Furthermore, in other embodiments,preservatives such as benzyl alcohol, phenol, sorbic acid, parabens, andchlorocresol are added.

Routes of Administration

Injectable formulations are administered using any method known in theart, for example, using a single needle, multiple needles, and/or usinga needleless injection device. In some embodiments, a tissue loadingdose of the active ingredients formulated in a suitable carrierdelivered by injection. In some embodiments, delivery comprises singleneedle injection. In some embodiments, delivery comprises injectionusing a multi-needle array, which, in some embodiments, provides a widedispersion of the formulation in the target tissue. In some embodiments,formulations are injected in a manner that allows dispersal into theappropriate layer of subcutaneous fat in or near regional fat areas.

Transcutaneous formulations, also contemplated as a route of deliveryfor the pharmaceutical formulations and methods of treatment providedherein, are administered using any known method in the art.

In some embodiments, the beta-2 agonist and the compound that reducesdesensitization are administered in a non-inhalation manner, for exampleby injection. In some embodiments, the formulations provided herein areadministered as separate formulations, or, alternatively, areadministered by separate routes administered followed by injection of alipophilic, long-acting beta-2 agonist. In some embodiments, thecompound that reduces desensitization is administered prior to thebeta-2 agonist. In other embodiments, the beta-2 agonist is administeredprior to the compound that reduces desensitization.

Embodiments of the composition are formulated for administered by anysuitable method, for example, as described in Remington: The Science AndPractice Of Pharmacy (21st ed., Lippincott Williams & Wilkins).Exemplary routes of administration include, but are not limited toparenteral, subcutaneous, or intramuscular. In some embodiments, thecomposition is formulated for injection of an area at which treatment isdesired, for example, in or near a regional fat deposit.

Any suitable pharmaceutically acceptable excipient appropriate for aparticular route of administration can be used. Examples ofpharmaceutically acceptable carriers include, but are not limited to,buffers, saline, or other aqueous media. The compounds described hereinare in some embodiments, soluble in the carrier which is employed fortheir administration (e.g., subcutaneous). Some embodiments comprise anysuitable lipophilic carrier, for example, modified oils (e.g.,Cremophor® BASF, Germany), soybean oil, propylene glycol, polyethyleneglycol, derivatized polyethers, combinations thereof, and the like. Someembodiments comprise one or more carriers or agents, suitable forsubcutaneous administration. Some embodiments comprise excipientssuitable for stable suspensions for beta-2 receptor agonists andglucocorticosteroids.

In some embodiments, the pharmaceutical formulations comprisepolyethylene glycol in an amount of from about 0.5% to about 40%. Inanother embodiment, the formulation suitable for subcutaneousadministration comprises polyethylene glycol in an amount from about0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, and about 40%. In afurther embodiment, polyethylene glycol is in an amount of about 20%. Inyet a further embodiment, polyethylene glycol is PEG 400. In yet anotherembodiment, the pharmaceutical formulations comprise polysorbate in anamount of from 0.01% to about 10%. In another embodiment, theformulation suitable for subcutaneous administration comprisespolysorbate in an amount from about 0.01%, about 0.02%, about 0.03%,about 0.04%, about 0.05%, about, about 0.06%, about 0.07%, about 0.08%,about 0.09%, 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%,about, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, andabout 9%. In a further embodiment, polysorbate is in an amount of about10%. In yet a further embodiment, polysorbate is polysorbate 80.

In other embodiments, another delivery mode comprises a needlesspressurized injection device. In some embodiments, of these devices, theformulation is pressurized mechanically or pneumatically, for example,using a gas such as helium or carbon dioxide, and then forced through asmall orifice into the body tissues, thereby delivering the formulationsubcutaneously. Suitable formulations for needless injections are known,for example, liquid, solutions, suspensions, gels, colloids, emulsions,and dry powders. An advantage of this system is a wide dispersal areacompared with typical needle injection systems. Needless injection underthe appropriate pressure forces the formulation into a more planardelivery pattern, with fingers of formulation spreading out radiallyfollowing paths of least resistance. In contrast, delivery by a typicalneedle injection results in a globular delivery of the formulation.Needless injection also permits precise control of the depth ofpenetration by controlling the injection pressure and orifice size.Thus, needless injection is a delivery method for a sub-dermal injectioncontemplated herein of a formulation for treating superficial fataccumulations, which is useful, for example, for smoothing skin dimplingcaused by fat. In other embodiments, needless injection is also used fordeeper fat accumulations. In further embodiments, a needless device alsoprovides easy and convenient multiple injections of the formulation overa defined region with a large lateral spread.

In some embodiments, the interval between administration of the compoundthat reduces desensitization and administration of the beta-2 agonistcan be an interval from less than about 5 minutes, about 5 minutes toabout 1 month, e.g., 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1hour, 6 hours, 12 hours, one day, 2 day, 3 days, 4 days, 5 days, 6 days,7 days, 10 days, 2 weeks, 3 weeks, or any other time interval from about5 minutes to about 1 month. In another embodiment, the compound thatreduces desensitization (e.g., a glucocorticosteroid) is administeredorally up to about 7 days, e.g., 3 days, 4 days, 5 days, 6 days, 7 days,8 days, 9 days, or 10 days prior to administering the beta-2 agonist(e.g., by local injection into or near a fat deposit).

In other embodiments, the beta-2 agonist is co-administeredconcomitantly (e.g., as part of the same formulation) with the compoundthat reduces beta-adrenergic receptor desensitization (e.g., aglucocorticosteroid). In further or additional embodiments,co-administration is provided sequentially (e.g., separately but oneagent administered after the other), or concurrently (e.g., at the sametime but the agents are, e.g., in different formulations).

In some embodiments, the subject to be treated is provided anon-sustained release formulation. In some embodiments, thenon-sustained release formulation, after a single dose, providesactivity of one or more long-acting selective beta-2 agonists for aduration from about 4 hours to about 24 hours, e.g., about 6 hours, 8hours, 10 hours, 12 hours, 16 hours, 18 hours, 21 hours, or any otherduration of beta-2 agonist activity from about four hours to about 24hours.

Treatment of Other Conditions

In some embodiments, the above-mentioned drugs and combinations are usedfor treating immune and inflammation-related dermal conditions includingpsoriasis, atopic dermatitis, vitiligo, hypopigmentation, stria, andwrinkles or rhytids. In one embodiment, a combination of lipophilic,long-acting selective beta-2 agonists and ketotifen is administeredsubcutaneously. In another embodiment, a combination of a beta agonistand a glucocorticoid are used for treating immune andinflammation-related dermal conditions. The principle of upregulatingthe beta-adrenergic receptor number and reducing the desensitization ofthe beta-adrenergic receptor through the use of ketotifen (or aglucocorticoid) in combination with a selective beta-2 agonist, such assalmeterol is useful in the treatment of dermal conditions such asdermatitis (e.g., atopic) and psoriasis. Other drugs which stabilize thebeta-2 adrenergic receptor and/or up regulate the receptor for treatingdermal conditions include, but are not limited to, thyroid hormone,1,25-dihydroxy vitamin D3 or it analogue, and bioflavanoids such asquercetin or fisetin. In some embodiments, darkening of skin orhypopigmented areas, such as occurs in vitiligo, stria, or a lack ofsunlight is treated with the proposed combinations and principles ofbeta-adrenergic receptor upregulation as described herein. In oneembodiment, a combination of long-acting beta-adrenergic agonists withother drugs that stabilize and/or increase the beta-2 adrenergicreceptor is used in a subcutaneous formulation to treat theafore-mentioned conditions. In some embodiments, a combination oflong-acting beta-adrenergic agonists with other drugs that enhance thecAMP response to beta-2 adrenergic receptor stimulation is utilized. Inone embodiment, a combination of salmeterol with ketotifen is used totreat psoriasis. In some embodiments, where the beta-2 adrenergicreceptor has defective activity, forskolin is used in the combination.Antigen presentation by the Langerhans cells may incite the immunecomponent of the disease. The pathogenesis of psoriasis includesoverproliferation of keratinocytes and immune inflammatory reactions,including lymphocyte (such as T-cells) migration and activation in thepsoriatic lesion. Psoriasis may be characterized by T-helper 1 (Th1)type responses. In some embodiments, subcutaneously administrablelipophilic, long-acting beta-adrenergic agonists are used to control theproliferation of the keratinocyte and lymphocytes including T-cells. Insome embodiments, long-acting beta-adrenergic agonists are also used toinhibit Th1 responses. In some embodiments, subcutaneously administrablelipophilic, long-acting beta-adrenergic agonist treatment is used todecrease Langerhans cell migration and antigen presentation. In someembodiments, ketotifen can be used to stabilize and upregulate beta-2adrenergic receptors on lymphocytes, keratinocytes, or dermalfibroblasts. In addition ketotifen inhibits the release of cytokinessuch as tumor necrosis factor alpha (TNF-alpha). TNF-alpha plays a rolein the pathogenesis of psoriasis. Thus, blocking the action of thiscytokine (e.g., with antibodies), or reducing the release of thecytokine reduces the severity of skin lesions. In some embodiments,ketotifen is administered to inhibit T-cell activity and thereby reduceinflammatory immune responses.

In another embodiment, long-acting beta-adrenergic agonists, such assalmeterol xinafoate, its stereoisomers, as physiologic salts, opticalisomers, racemates, solvates or polymorphs thereof, are combined with1,25-dihydroxy Vitamin D3 or its analogues. In some embodiments,1,25-dihydroxy Vitamin D3 enhances beta-adrenergic adenylate cyclaseresponses in keratinocytes. In conditions, such as psoriasis, cAMPlevels are low or cAMP formation is impaired. Accordingly, in someembodiments, where a subject is suffering from psoriasis, Vitamin D3 isadministered in combination with a subcutaneous formulation consistingessentially of a long-acting beta-adrenergic agonist, such assalmeterol.

In some embodiments, beta-2 adrenergic receptor agonists areadministered subcutaneously for the treatment of skin wrinkles and skinstria, or stretch marks. Cutaneous stria are characterized by a thinningof the dermis, with a loss of collagen and hypopigmention. Long-actingbeta-adrenergic agonists promote the recruitment and proliferation andcollagen production of dermal fibroblasts in the stria. In addition,they stimulate melanocytes to repigment the stria. Thus, in someembodiments a subcutaneous formulation consisting essentially of anadipose tissue-reducing amount of salmeterol or a salt, solvate, orpolymorph thereof is used in combination with other drugs to stabilizeand up regulate the beta-2 adrenergic receptor such as those disclosedabove, including ketotifen, glucocorticoids, thyroid hormone, andbioflavanoids quercetin and fisetin. In some embodiments, forskolin isused with the lipophilic, long-acting beta-adrenergic agonist and incombination with the previously disclosed compounds (e.g. quercetin,fisetin, glucocorticoid, or ketotifen) to treat cutaneous stria andwrinkles.

The combination of a selective lipophilic long-acting beta-adrenergicagonist is also suitable for the treatment of cachexia. Ketotifen cancause weight gain, which may be due to increased food intake secondaryto appetite or satiety effects. Hence, the combination may havepronounced effects on cachexia, or wasting syndromes, secondary to othermedical conditions such as HIV infection, cancer, or heart failure.Additionally, as previously disclosed the combination of a long-actingbeta-2 adrenergic receptor agonist and ketotifen in a formulationsuitable for parenteral administration, such as subcutaneousadministration, has enhanced effects by increasing beta-adrenergicreceptor numbers and reducing receptor deactivation.

In some embodiments, beta-2 adrenergic receptor agonists areadministered to increase skeletal muscle mass and cause hypertrophy andincreased protein synthesis, effects which are mediated throughintracellular in increases cAMP levels. Similar to adipocytes, exposureto beta-2 adrenergic receptor agonists results in receptordown-regulation. Thus, in the disclosed formulations, combinations arealso used for treating skeletal muscle injury or conditions whereincreasing skeletal muscle mass is important. In some embodiments, themethods described herein are used to increase facial muscle tone andprovide a more youthful appearance. In some embodiments, the methodsdescribed herein are used to treat strabismus or lazy eye bystrengthening ocular muscles. In some embodiments, combinations includeadipose tissue-reducing lipophilic, long-acting beta-adrenergicagonists, such as salmeterol xinafoate, and compounds that reducedesensitization of beta-2 adrenergic receptors, e.g., beta-2 adrenergicreceptor stabilizers/upregulators glucocorticoids or ketotifen in asuitable formulation for subcutaneous administration. In someembodiments, bioflavanoids, such as quercetin or fisetin, are also usedto decrease beta-2 adrenergic receptor desensitization. In someembodiments, glucocorticoids and adipose tissue-reducing lipophilic,long-acting beta-adrenergic agonists are co-administered to a subject torepair a muscle injury. In some embodiments, agents that increase cAMPsuch as forskolin are used for treating skeletal muscle injury orimproving facial muscle tone.

In other embodiments, provided are methods for decreasing cellulite in asubject, also known as adiposis edematosa, dermopanniculosis deformans,status protrusus cutis, and gynoid lipodystrophy, comprisingadministering via subcutaneous methods, a subcutaneous formulationconsisting essentially of a long-acting beta-2 adrenergic receptoragonist and a subcutaneously acceptable excipient thereof, wherein theformulation decreases cellulite in the subject.

Areas of fat deposits on a subject, such as for example a human patient,for which the formulations described herein are useful include, but arenot limited to, the inside region of the knees, the middle to upper areaof the upper arm, including the tricep area, the submental area,including the area under the chin, for example the wattle (which isunderstood to refer to the fleshy fold of skin in the submental area ofa patient), the abdomen, the hips, the inner thigh, the outer thigh, thebuttocks, the lower back and the chest.

In some embodiments, inducing lipolysis and inhibiting fat cell growthin regional fat accumulations, have additional health benefits throughthe shrinkage of the average fat cell diameter or volume. Large volumefat cells actively secrete pro-inflammatory and deleterious hormonessuch as TNF-alpha and interleukins (“adipokines”), which contribute tocomorbidities associated with fat, such as diabetes. By reducing thesize of these fat cells and therefore the deleterious adipokinesecretion, improvements in fat-related comorbidities are realized.

In some embodiments, the disclosed formulations (e.g., subcutaneous andtranscutaneous formulations), and combinations described herein are usedfor treating obstructive sleep apnea. Obstructive sleep apnea occurswhen the airway is temporarily blocked during sleep, leading to hypoxia,high blood pressure, cardiac dysrhythmia, and a higher risk of death.Excessive fat in the pharynx and soft palate it believed to contributeto this blockage. Obese people have a higher incidence of sleepdisorders and persons with sleep apnea have excessive fat in the palateand pharynx on MRI. Thus, in some embodiments, formulations describedare administered to a subject to reduce the symptoms of sleep apnea. Insome embodiments, the formulations are administered locally (e.g., byinjection) into the palate or pharynx transorally. In some embodiments,the formulations are administered by subcutaneous into the region theneck to reduce the obstructive symptoms.

While certain embodiments have been described, these embodiments havebeen presented by way of example only, and are not intended to limit thescope of the disclosure. The formulations, methods, and systemsdescribed herein may be embodied in a variety of other forms.Furthermore, various omissions, substitutions and changes in the form ofthe formulations, methods, and systems described herein may be madewithout departing from the spirit of this disclosure. The accompanyingclaims and their equivalents are intended to cover such forms ormodifications.

EXAMPLES

The following specific examples are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. The examples described herein reference and providenon-limiting support to the various embodiments described in thepreceding sections.

Example 1 Plasma Pharmacokinetics Following Single Intravenous andSubcutaneous Injections of Salmeterol Xinafoate

Materials and Methods:

Salmeterol xinafoate was formulated in 5% PEG-400 in 0.9% saline, USP,at concentrations of 0.1, 1, 10, and 100 μg/mL. 1 male and 1 femalenon-naive Gottingen minipigs (18.4 to 19.5 kg at the time of the IVdose) were utilized. The animals were housed individually. The dosingscheme is shown in Table 1 below:

Injection # Route Dose (μg/kg) No. Animals/sex 1 IV 4 1 2 SC 4 1 3 SC* 41 4 SC* 0.004 1 5 SC* 0.04 1 6 SC* 0.4 1 *The dose volume was equallydivided over 5 injection sites

Each minipig received a single IV injection followed by SC doses of thesalmeterol formulation. The IV dose was administered by slow bolus (1minute; 0.0167 hr) into the marginal ear vein, whereas the first SC dosewas administered as a bolus injection along the back (neck-region) wherethere was a longitudinal fat depot. The second SC dose was distributedin 5 separate SC injection sites. The fourth, fifth, and sixthadministrations were SC administrations divided over 5 injection sits ofdifferent graded concentrations (0.1, 1, and 10 μg/mL of salmeterol in5% PEG). Each dose was separated by at least a 3-day washout period.Blood samples (approximately 4 mL) were collected via thebranchiocephalic plexus at pre-dose and at 2.00, 5.00, 10.0, 20.0, 30.0and 45.0 minutes, and at 1.00, 2.00, 4.00, 8.00 and 24.0 hr post-dose.Blood samples were placed in tubes containing K₂-EDTA as theanticoagulant. Samples were processed to plasma by centrifugation andstored frozen at approximately −70° C. (+/−15° C.) until shipment foranalysis.

Sample Analysis:

Plasma samples were analyzed for salmeterol using a qualified liquidchromatography/mass spectrometry/mass mass spectrometry (LC/MS/MS)method. The lower limit of quantitation (LLOQ) was 2.50 pg/mL. Sampleswere analyzed within a maximum of 14.0 days of collection.

Data Analysis:

Non-compartmental pharmacokinetic parameters were calculated usingWinNonlin 5.2 software, NCA model 202, IV infusion input for the IV dataand NCA model 200, extravascular input for the SC data. Individualplasma concentrations for each animal were used in the calculation ofpharmacokinetic parameters. Nominal collection times and doses were usedin the calculations. The area under the plasma concentration-time curve(AUC) was calculated using linear trapezoidal approximation (linear/loginterpolation). Concentration values below the assay limit ofquantitation were set to zero for calculations. The maximum plasmaconcentration (C_(max)) and the time of its occurrence (T_(max)) wereverified by inspection. The half-life (t_(1/2)) values were calculatedusing the last 3 plasma concentrations with nonzero values, if datapermitted.

Results:

The pharmacokinetic parameters are shown in Table 2. The plasmaconcentrations of salmeterol are shown in Table 3. The plasmaconcentrations of salmeterol and formoterol (via subcutaneous andintravenous administration) are shown in FIG. 1. Actual sampling timesdiffered from nominal sampling times on occasion by more than 10%. Datain the apparent elimination phase were inadequate to calculate somepharmacokinetic parameters for some animals (indicated as ND for notdetermined or NA for not applicable). Bioavailability was likely poorlyestimated due to scarcity of data in the apparent terminal eliminationphase to calculate the apparent terminal elimination rate constant. Thisaffected calculation of AUC_(inf), t_(1/2), CL, V_(ss), and F. Thesedepartures are not believed to have significantly impacted the overallpharmacokinetic conclusions.

TABLE 2 Pharmacokinetic Parameters of Salmeterol Following IV and SCAdministration to Gottingen Minipigs T_(max) C_(max) AUC_(inf) t_(1/2)t_(last) CL V_(ss) Route Sex Animal (hr) (Pg/mL) (pg · hr/mL) (hr) (hr)(mL/hr/kg) (mL/kg) IV Female 2-0623 0.0333 4950 1470 9.52 24.0 273022100 IV Male 1-0771 0.0333 4290 1750 7.89 24.0 2280 17100 Dose T_(max)C_(max) AUC_(inf) t_(1/2) t_(last) Route (μg/kg) Sex Animal (hr) (Pg/mL)(pg · hr/mL) (hr) (hr) F SC1 4 Female 2-0623 1.00 403 1850 6.02 24.01.26 SC5 4 Female 2-0623 0.0333 626 2010 8.58 24.0 1.37 SC1 4 Male1-0771 0.0833 575 1290 5.92 24.0 0.737 SC5 4 Male 1-0771 0.0833 681 15106.84 24.0 0.863 SC5 0.004 Female 2-0623 1.00 3.08 ND ND 1.00 ND SC50.004 Male 1-0771 NA 0 ND ND NA ND SC5 0.04 Female 2-0623 0.0333 8.31 NDND 0.0833 ND SC5 0.04 Male 1-0771 0.0833 2.77 ND ND 0.0833 ND SC5 0.4Female 2-0623 0.0333 113 164 2.88 8.00 1.12 SC5 0.4 Male 1-0771 0.083364.5 138 2.94 8.00 0.789 AUC_(inf) = area under the curve at infinitetime. CL = plasma clearance. F = bioavailability; NA = not applicable;ND = not determined; SC1—single site injection; SC5 = SC injection splitamong 5 sites. t_(last) = time of last measurable plasma concentration.V_(ss) = volume of distribution at steady state The IV dose was 4 μg/kgThe injection 4 SC5 0.004 μg/kg male showed no plasmaconcentrations >LLOQ of salmeterol at any time point.

TABLE 3 Plasma Concentrations of Salmeterol Following IV and SCAdministration to Gottingen Minipigs (08-529) Dose Time (hr) Inj Rte(mg/kg) Sx 0 0.0333 0.0833 0.167 0.333 0.500 0.750 1.00 2.00 4.00 8.0024.0 1 IV 4 M < 4290 1380 502 326 240 177 161 121 70.6 53.6 12.5 1 IV 4F < 4950 1300 559 322 232 152 154 81.1 49.1 35.3 11.3 2 SC 4 M < 495 575423 284 278 236 212 168 74.5 30.6 6.23 2 SC 4 F < 124 227 206 334 302351 403 206 108 54.4 10.0 3 SC5 4 M < 604 681 519 449 483 278 251 14875.6 36.1 8.92 3 SC5 4 F < 626 485 442 398 435 435 388 196 86.8 51.016.1 4 SC5 0.004 M < < < < < < < < < < < < 4 SC5 0.004 F < < < < < < <3.08 < < < < 5 SC5 0.04 M < < 2.77 < < < < < < < < < 5 SC5 0.04 F < 8.313.17 < < < < < < < < < 6 SC5 0.4 M < 32.0 64.5 57.0 43.3 53.8 26.5 28.719.5 8.46 4.41 < 6 SC5 0.4 F < 113 55.4 42.9 59.8 83.5 34.5 32.2 24.58.58 5.15 < Plasma concentrations are reported in pg/mL. LLOQ = 2.50pg/mL < = LLOQ; F = female; Inj = Injection number; M = male; Rte =route; SC1 = single site injection; SC5 = SC injection split among 5sites; Sx = sex. The male animal was number 1-0771 and the female animalnumber was number 2-0623

Overall there were no consistent, substantial differences in C_(max) orAUC_(inf) values between the male and female animal. Additionally, therewas not a substantial difference in C_(max) and AUC_(inf) values betweensingle site SC doses and multiple (5) site SC doses at the 4 μg/kg dose.The ratios (SC1/SC5) ranged from 0.644 to 0.844 for C_(max) and 0.854and 0.920 for AUC_(inf). Salmeterol appeared to be well absorbed afterSC administration at a single site at 4 μg/kg and after divided SCadministration at 5 sites at the 4 and 0.4 μg/kg doses; bioavailabilityranged from 0.737 to 0.863 for the male and 1.26 to 1.37 for the femalefor the 4 μg/kg dose regardless of the number of sites of administrationand 0.789 for the male and 1.12 for the female for the 0.4 μg/kg dose at5 divided SC sites. Scarce or no plasma concentrations were observed forthe 0.04 and 0.004 divided SC doses.

Conclusion:

In this single male and female Gottingen minipig study, salmeterolappeared to be well absorbed after subcutaneous administration at asingle site and after divided SC administration at 5 sites at the 4μg/kg dose and after divided SC administration of the 0.4 μg/kg dose.Overall there were no consistent, substantial differences in Cmax andAUC_(inf) values between single site SC doses vs. multiple site SCdoses. Overall there were no substantial differences in pharmacokineticparameters between the male and female animals.

Example 2 Plasma Pharmacokinetics Following a Single Intravenous andSubcutaneous Injection of a Combination of Formoterol and Budesonide inGottingen Minipigs

Materials and Methods:

Formoterol fumarate dehydrate and budesonide were formulated in 2%PEG-400 in saline. 1 male and 1 female non-naive Gottingen minipigs (8to 12 kg upon receipt per protocol) were utilized. The animals werehoused individually. The dosing scheme is shown in Table 4 below:

Dose (μg/kg) Injection # Route Budesonide Formoterol No. Animals/sex 1IV 1.6 0.4 1 2 SC 1.6 0.4 1 3 SC* 1.6 0.4 1 *The dose volume was equallydivided over 5 injection sites

Each minipig received a single IV injection of the combined formoteroland budesonide formulation followed by testing of the SC doses. Eachdose was separated by at least a 3-day washout period. The IV dose wasadministered by slow bolus (1 minute; 0.017 hr) into the marginal earvein, whereas the first SC dose was administered as a bolus injectionalong the back (neck-region) where there was a longitudinal fat depot.The second SC dose was distributed in 5 separate proximal SC injectionsseparated by approximately 2 cm. Blood samples (approximately 4 mL) werecollected via the branchiocephalic plexus at pre-dose and at 2, 5, 10,20, 30 and 45 minutes, and at 1, 2, 4, 8 and 24 hr post-dose. Bloodsamples were placed in tubes containing K₂-EDTA as the anticoagulant.Samples were processed to plasma by centrifugation and stored frozen atapproximately −70° C. (+/−15° C.) until shipment for analysis.

Sample Analysis:

Plasma samples were analyzed for formoterol and budesonide using aqualified liquid chromatography/mass spectrometry/mass mass spectrometry(LC/MS/MS) method. The lower limit of quantitation (LLOQ) was 1.00 pg/mLfor formoterol and 25.0 pg/mL for budesonide.

Data Analysis:

Non-compartmental pharmacokinetic parameters were calculated usingWinNonlin 5.2 software, NCA model 202, IV infusion input for the IV dataand NCA model 200, extravascular input for the SC data. Individualplasma concentrations for each animal were used in the calculation ofpharmacokinetic parameters. Nominal collection times and doses were usedin the calculations. The area under the plasma concentration-time curve(AUC) was calculated using linear trapezoidal approximation (linear/loginterpolation). Concentration values below the assay limit ofquantitation were set to zero for calculations. The maximum plasmaconcentration (C_(max)) and the time of its occurrence (T_(max)) wereverified by inspection. The half-life (t_(1/2)) values were calculatedusing the last 3 plasma concentrations with nonzero values, if datapermitted.

Results:

The pharmacokinetic parameters are shown in Table 5. The plasmaconcentrations of formoterol and budesonide are shown in Table 6 andFIG. 1. The pre-dose period 1 IV sample of male animal 1-0771 showed alow, but measurable budesonide concentration and the pre-dose period 1IV sample of female animal 2-0623 showed a low, but measurableformoterol concentration. Other occasional measurable pre-doseconcentrations of formoterol or budesonide are explainable as carryoverfrom previous doses. Actual sampling times differed from nominalsampling times on occasion by more than 10%. Data in the apparentelimination phase were inadequate to estimate the t_(1/2) for the maleanimal 1-0771 for the SC1 group for budesonide.

TABLE 5 Pharmacokinetic Parameters for Formoterol and BudesonideFollowing IV and SC Administration of the Combination to GottingenMinipigs (08-522) T_(max) C_(max) AUC_(inf) t_(1/2) t_(last) CL V_(ss)Analyte Route Sex Animal (hr) (pg/mL) (pg · hr/mL) (hr) (hr) (mL/hr/kg)(mL/kg) Formoterol IV Male 1-0771 0.0333 410 264 2.54 8.00 1520 3940Formoterol IV Female 2-0623 0.0333 900 328 2.86 8.00 1220 3170Budesonide IV Male 1-0771 0.0333 760 781 2.73 4.00 2050 6290 BudesonideIV Female 2-0623 0.0333 1350 764 1.61 4.00 2090 3380 AUC_(inf) AnalyteRoute Sex Animal T_(max) (hr) C_(max) (pg/mL) (pg · hr/mL) t_(1/2) (hr)t_(last) (hr) F Formoterol SC1 Male 1-0771 0.0333 378 627 5.51 24.0 2.38Formoterol SC5 Male 1-0771 0.167 217 579 4.19 24.0 2.19 Formoterol SC1Female 2-0623 0.0833 284 378 2.33 8.00 1.15 Formoterol SC5 Female 2-06230.0833 256 577 12.8 24.0 1.76 Budesonide SC1 Male 1-0771 0.0833 979 202018.8 24.0 2.59 Budesonide SC5 Male 1-0771 0.0833 1060 981 1.01 4.00 1.26Budesonide SC1 Female 2-0623 0.167 798 992 1.41 4.00 1.30 Budesonide SC5Female 2-0623 0.333 697 884 1.03 4.00 1.16 AUC_(inf) = area under thecurve at infinite time. CL = plasma clearance. F = bioavailability. SC1= single site SC injection; SC5 = SC injection split among 5 sites.t_(last) = time of last measurable plasma concentration. V_(ss) = volumeof distribution at steady state.

TABLE 6 Plasma Concentrations of Formoterol and Budesonide Following IVand SC Administration of the Combination to Gottingen Minipigs (08-522)Time (hr) Route Animal Sex 0 0.0333 0.0833 0.167 0.333 0.500 0.750 1.002.00 4.00 8.00 24.0 Formoterol IV 1-0771 Male <LLOQ 410 99.6 90.2 68.957.0 48.8 81.3 26.5 18.3 5.33 <LLOQ SC1 1-0771 Male 4.63 378 294 147 121161 79.0 83.3 82.2 42.2 15.2 2.87 SC5 1-0771 Male <LLOQ 133 216 217 15093.8 94.1 80.1 71.1 35.9 18.1 1.30 IV 2-0623 Female 3.53 900 204 11073.5 98.4 61.4 45.4 31.3 18.4 7.26 <LLOQ SC1 2-0623 Female <LLOQ 169 284237 156 127 78.9 67.8 51.2 28.5 8.58 <LLOQ SC5 2-0623 Female <LLOQ 94.6256 178 131 100 82.6 62.8 44.4 26.0 10.3 6.95 Budesonide IV 1-0771 Male41.9  760 539 499 356 308 204 138 60.5 57.5 <LLOQ <LLOQ SC1 1-0771 Male<LLOQ 806 979 903 703 466 285 222 97.9 43.2 <LLOQ 31.6  SC5 1-0771 Male<LLOQ 902 1060 933 647 538 438 317 92.4 36.5 <LLOQ <LLOQ IV 2-0623Female <LLOQ 1350 939 592 335 307 285 169 69.8 42.4 <LLOQ <LLOQ SC12-0623 Female <LLOQ 250 559 798 784 598 431 238 117 51.9 <LLOQ <LLOQ SC52-0623 Female <LLOQ 249 637 434 697 535 382 328 100 39.5 <LLOQ <LLOQPlasma concentrations are reported in pg/mL. LLOQ = 1.00 pg/mL forformoterol and 25.0 pg/mL for budesonide SC1 = single site SCinjections; SC5 = SC injection split among 5 sites.

Overall there were no consistent, substantial differences in C_(max) orAUC_(inf) values between the male and female animal for formoterol orbudesonide. Additionally, there were no consistent, substantialdifferences in C_(max) and AUC_(inf) values for formoterol or budesonidebetween single site SC doses and multiple (5) site SC doses. Bothformoterol and budesonide appeared to be well absorbed after SCadministration at a single site and after divided SC administration at 5sites; however, bioavailability was likely poorly estimated due toscarcity of data in the apparent terminal elimination phase to calculatethe apparent terminal elimination rate constant This affectedcalculation of AUC_(inf), t_(1/2), CL, V_(ss), and F.

Conclusion:

In this single male and female Gottingen minipig study, both formoteroland budesonide appeared to be well absorbed after subcutaneousadministration at a single site and after divided SC administration at 5sites at the 4 μg/kg dose and after divided SC administration of the 0.4μg/kg dose. Overall there were no consistent, substantial differences inCmax and AUC_(inf) values between single site SC doses vs. multiple siteSC doses. Overall there were no substantial differences inpharmacokinetic parameters between the male and female animals.

Example 3 Clinical Trials Example 3A Phase 1—Open-Label Evaluation ofthe Pharmacokinetics and Safety of Salmeterol Xinafoate and FluticasonePropionate Co-Administered Subcutaneously in Healthy Volunteers

Objectives:

To evaluate the pharmacokinetics and safety of increasing single dosesof salmeterol xinafoate (SX) administered by subcutaneous injection tohealthy volunteers (HV). To also evaluate the pharmacokinetics andsafety of a single dose of fluticasone propionate (FP) administered bysubcutaneous injection to healthy volunteers whereby the single dose ofFP yields a mean systemic C_(max) not exceeding 100 pg/mL. The objectiveof this study is to also evaluate the pharmacokinetics and safety of asingle dose of the selected SX dose and selected FP dose co-administeredas a single subcutaneous injection to healthy volunteers. Additionally,the objective of this study is to evaluate the pharmacokinetics andsafety following multiple doses of the selected SX+FP combined doseadministered by subcutaneous injection to healthy volunteers (either 1time per week for 4 weeks or 3 times per week for 4 weeks).

Study Design:

Part I: An open-label, sequential dose-escalation pharmacokinetic studyof single doses of SX administered subcutaneously in ten (10) healthyvolunteers. Part II: An open-label, fixed sequence, cross-overpharmacokinetic study to investigate potential interactions between theselected FP dose and the selected SX dose administered subcutaneously ineight (8) healthy volunteers. Part III: An open-label, multiple-dosepharmacokinetic study of the selected SX+FP combined dose administeredsubcutaneously either 1 time per week for 4 weeks or 3 times per weekfor 4 weeks in twelve (12) healthy volunteers.

Study Drug:

A 400 μg/mL sterile, preservative-free, clear, aqueous solution forinjection of SX is contained in a 5 mL single-use glass vial. Each 5 mLsingle-use glass vial contains 2.6 mL of SX solution (400 μg/mL) whichis stored frozen (−15° C. to −25° C.) and protected from light untildose preparation. A 25 mg/mL sterile, preservative free, clear, aqueoussolution for injection of FP is contained in a 2 mL single-use glassvial. Each 2 mL single-use glass vial contains 1.0 mL of FP solution (25μg/mL) which is stored frozen (−15° C. to −25° C.) and protected fromlight until dose preparation. Diluent: A sterile, preservative free,clear, aqueous solution of 20% PEG 400, 1% polysorbate 80, and sterilewater for injection contained in a 10 mL single-use glass vial. Each 10mL single-use glass vial contains 5.0 mL of diluent solution whichshould be stored at room temperature (15° C. to 25° C.). Prior toadministration, the drug products are thawed, mixed, and if necessarydiluted with sterile diluent to provide the target concentrations fordose administration. Study drug is subcutaneously administered tosubjects using a sterile, disposable single-use syringe with anappropriate gauge needle. Instructions for study drug storage,preparation, and administration comply with <USP> 797 guidance forCompounding Sterile Preparations.

Study Population:

Healthy male and female subjects, 18 to 55 years of age.

Inclusion Criteria:

-   -   a. Male and female subjects, 18 to 55 years of age, in good        health and free of any disease or physical condition which        might, in the investigator's opinion, expose the subject to an        unacceptable risk by study participation;    -   b. Written, informed consent prior to any study procedures        (including screening) being performed;    -   c. BMI≧18 kg/m² and <30 kg/m²;    -   d. Minimum 2 cm abdominal skin fold on vertical pinch 2 cm        lateral to umbilicus as determined by the site using        Accu-Measure calipers;    -   e. History of a stable exercise routine over the last 3 months        or a sedentary lifestyle (less than 60 minutes of exercise per        week);    -   f. Agreement to adhere to the study requirements and        restrictions including all required assessments and visits.

Exclusion Criteria:

-   -   a. Women of childbearing potential who are pregnant, lactating,        and/or who are not using adequate birth control methods;    -   b. Known hypersensitivity to the study drugs or any of their        components;    -   c. Deemed by the investigator to be unreliable and unlikely to        comply with protocol procedures or adhere to the study visit        schedule;    -   d. Any medical condition that in the opinion of the investigator        might jeopardize the subject's safety or interfere with the        absorption, distribution, metabolism, or excretion of the study        drug, including:        -   i. Major surgery within 30 days prior to day 0 or planned            surgery during the study period;        -   ii. Seropositive for HIV, hepatitis B surface antigen or            hepatitis C, or previously diagnosed disease(s) of immune            function where absolute neutrophil count <1000 mm³;        -   iii. Any clinically significant abnormal laboratory, ECG            (including cardiac arrhythmia) and/or physical exam findings            during screening and/or day 0;        -   iv. Skin disease or any other condition which is the opinion            of the investigator might interfere with clinical assessment            of the injection site area at screening or day 1;        -   v. Hemoglobin level below 12 g/dL at screening;        -   vi. History of diabetes mellitus or cardiovascular disease            (subjects with well-controlled hypertension will not be            excluded);        -   vii. A consistently abnormal pulse rate, abnormal resting            supine blood pressure, and/or a predisposition to            orthostatic hypotension if considered clinically            significant;    -   e. Recent history of drug or alcohol abuse within 90 days prior        to screening;    -   f. Use of any tobacco products within 90 days prior to        screening;    -   g. Donation of blood/plasma and/or any significant blood loss        for any reason greater than 450 mL with 60 days prior to        screening;    -   h. Use of tricyclic antidepressants or monoamine oxidase        inhibitors medications within 14 days prior to day 0 or during        the study;    -   i. Other exclusion criteria such as use of other systemic        prescription medications, use of any oral herbal or dietary        supplement, use of alcohol-, caffeine-, or xanthine-containing        products.

Pharmacokinetic Parameters:

The following parameters will be measured/determined: AUC_(0-t),AUC_(0-inf), C_(max), T_(max), k_(el), A_(UCt/inf), and t_(1/2el).

Results—Single Dose Pharmacokinetics:

FIG. 2A shows the plasma concentration of 2 doses of salmeterolxinafoate (19 μg and 52 μg) following a 1 mL subcutaneous injection intoabdominal fat of a group of 10 subjects. Plasma concentration ofsalmeterol xinafoate (52 μg) following a 1 mL subcutaneous injectioninto abdominal fat of a second group of 8 subjects is also shown in FIG.2A. This group of subjects also received a 1 mL subcutaneous injectionof fluticasone propionate (22 μg) (FIG. 2B). Finally, this group of 8subjects received a 1 mL subcutaneous injection of a combination ofsalmeterol xinafoate (52 μg) and fluticasone propionate (22 μg) intoabdominal fat; the plasma concentrations of each are shown in FIGS. 2Aand 2B (combo).

Results—Multiple Dose Pharmacokinetics:

FIG. 2A shows the pharmacokinetic profile for 52 μg of salmeterolxinafoate administered to the patient group that received 1 subcutaneousinjection per week for 4 consecutive weeks. FIG. 2B shows thepharmacokinetic profile for 22 μg of fluticasone propionate administeredto the patient group that received 1 subcutaneous injection per week for4 consecutive weeks. The profiles demonstrate that the systemic exposurelimits for salmeterol xinafoate and fluticasone propionate will notexceed those of the commercially available ADVAIR DISKUS® 500/50 drugproduct. Each profile depicted in FIGS. 2A and 2B demonstrates anincrease in Cmax and AUC at day 22 compared to day 1, which issuggestive of tissue remodeling (based on a reduced amount of adiposetissue at day 22).

Results—Safety Assessments:

No serious adverse events were observed in the patients studied.Specifically, no adverse cardiovascular events were observed and nosignificant skin changes (e.g., atrophy, pigmentation, nodularity, ornecrosis) were observed. Only minimal skin reactions (transient pain andirritation) were observed.

Example 3B Phase 2a—a Dose-Ranging Frequency Evaluation of the Safetyand Efficacy of Salmeterol Xinafoate and Fluticasone PropionateCo-Administered for Subcutaneous Injection for the Reduction ofAbdominal Subcutaneous Adipose Tissue

Objectives:

To evaluate in a single masked, placebo-controlled design, the safetyand exploratory efficacy measures of subcutaneous injections of threedifferent doses of the combination of salmeterol xinafoate (SX) andfluticasone propionate (FP) administered by subcutaneous injection 1 or2 times per week for 4 weeks to subjects with measurable abdominalsubcutaneous adipose tissue.

Study Design:

A single-masked, placebo-controlled multiple-dose study of the safetyand efficacy of three different doses of the combination of salmeterolxinafoate (SX) and fluticasone propionate (FP) administered bysubcutaneous injection 1 or 2 times per week for 4 weeks in 60 subjectswith measurable abdominal subcutaneous adipose tissue. The threeselected doses were chosen to yield a systemic exposure ranging fromapproximately 10-fold to 100-fold lower than the reference-listed ADVAIRDISKUS® 500/50 drug product.

Study Drug—Active Ingredient:

A 400 μg/mL sterile, preservative-free, clear, aqueous solution forinjection of SX is contained in a 5 mL single-use glass vial. Each 5 mLsingle-use glass vial contains 2.6 mL of SX solution (400 μg/mL) whichis stored frozen (−15° C. or below) and protected from light until dosepreparation. A 25 mg/mL sterile, preservative free, clear, aqueoussolution for injection of FP is contained in a 2 mL single-use glassvial. Each 2 mL clear glass vial contains 1.0 mL of FP solution (25μg/mL) which is stored frozen (−15° C. or below) and protected fromlight until dose preparation. Diluent: A sterile, preservative free,clear, aqueous solution of 20% PEG 400, 1% polysorbate 80, and sterilewater for injection contained in a 10 mL clear glass vial. Each 10 mLsingle-use glass vial contains 5.0 mL of diluent solution which shouldbe stored at room temperature (15° C. to 25° C.). Diluent or a versionof the Diluent further diluted with sterile water for injection (SWFI)served as the placebo.

The supplies are provided in nested bulk packaging. Prior toadministration, the drug products will be thawed, mixed, and ifnecessary, diluted under ascetic conditions with sterile Diluent orsterile water for injection to provide the target concentrations fordose administration. Study drug a placebo will be administeredsubcutaneously at the marked injection sites using a sterile, disposablesingle-use appropriately sized Luer Lock syringe with a 30 gauge 0.5inch needle.

Treatment Cohorts:

A single-masked, multiple-dose study of different doses and dosingfrequencies of SX and FP injected subcutaneously on one side of theabdomen 1 or 2 times per week for 4 weeks to subjects with measurableabdominal subcutaneous adipose tissue. Placebo injections on thecontra-lateral side of the abdomen will serve as control. The SX and FPdose will be administered as 1 mL injections 1 or 2 times per week tothe injection site, either on the right or left of the umbilicusaccording to randomization. The corresponding control injection site onthe contra-lateral side of the umbilicus will be injected with 1 mL ofplacebo in the identical sequence on the designated injection days. Thesequence of injections will continue until for 4 consecutive weeks for atotal of 4 or 8 subcutaneous injections of study drug and 4 or 8subcutaneous injections of placebo, depending on randomization.Comprehensive safety assessments will be performed at each subject visitthrough the end of the study at week 8. Eligible subjects will berandomized to one of the following six treatment cohorts (n=10 pergroup) for the 4 consecutive week treatment period.

1.0 μg FP and 0.5 μg SX and placebo injections once or twice per week

1.0 μg FP and 5.0 μg SX and placebo injections once or twice per week

1.0 μg FP and 10.0 μg SX and placebo injections once or twice per week

Duration of Treatment:

The study duration lasted 8 weeks: 4 week treatment period followed by 1week and 4 week post-therapy assessment follow-up visit for safety andefficacy.

Study Population:

Healthy male and female subjects, 18 to 55 years of age. A total numberof 60 subjects will be enrolled into 6 treatment cohorts. Subjects whoprovided informed consent in writing but who do not receive study drugfor any reason will be considered screen failures and will be replaced.

Inclusion Criteria:

-   -   a. Male and female subjects, 18 to 55 years of age, in good        health and free of any disease or physical condition which        might, in the investigator's opinion, expose the subject to an        unacceptable risk by study participation;    -   b. Anterior abdominal skin fold thickness of between 30 mm to 50        mm, inclusive, at 2 cm to the right and left of the umbilicus        measured with pinch calipers at screening day 0, and a        subcutaneous adipose tissue thickness of greater than or equal        to 2.5 cm and less than or equal to 5.0 cm on 2-dimensional        ultrasound at day 0;    -   c. BMI greater than or equal to 22 kg/m² and less than or equal        to 30 kg/m² and a history of stable weight in the last 3 months        at screening;    -   d. History of stable exercise routine over the last 3 months (at        least 120 minutes per week of moderate exercise, e.g., brisk        walking) at screening;    -   e. Written, informed consent prior to any study procedures        (including pre-treatment and screening) being performed;        agreement to adhere to the study requirements and restrictions        including all required assessments and visits.

Exclusion Criteria:

-   -   a. Women of childbearing potential who are pregnant, lactating,        and/or who are not using adequate birth control methods;    -   b. Known hypersensitivity to the study drugs or any of their        components;    -   c. Deemed by the investigator to be unreliable and unlikely to        comply with protocol procedures or adhere to the study visit        schedule;    -   d. Any medical condition that in the opinion of the investigator        might jeopardize the subject's safety or interfere with the        absorption, distribution, metabolism, or excretion of the study        drug, including:        -   i. Major surgery within 30 days prior to day 0 or planned            surgery during the study period;        -   ii. Any clinically significant abnormal laboratory result at            screening;        -   iii. Skin disease or any other conditions in the area of            injection sites at screening or day 0 which in the opinion            of the investigator might interfere with clinical assessment            of the injection site area;        -   iv. History of diabetes or cardiovascular disease (subjects            with well-controlled hypertension will not be excluded);        -   v. An abnormal pulse rate, abnormal resting supine blood            pressure, and/or a predisposition to orthostatic hypotension            if considered clinically significant;    -   e. Recent history of drug or alcohol abuse within 90 days prior        to screening;    -   f. Donation of blood/plasma and/or any significant blood loss        for any reason greater than 450 mL with 60 days prior to        screening (or during study period);    -   g. Prior enrollment in any SX and FP for injection study;    -   h. Concurrent enrollment in another investigational drug or        device study; or use of any experimental or investigational drug        or device within 30 days, or for drugs within 6 times the        elimination half-life prior to day 0 if that is longer;    -   i. Use of systemic, inhaled, or topical corticosteroids,        immunodulators, anti-metabolites, beta 2-andrenergic receptor        agonists, beta 2-andrenergic receptor blockers, or        nonpotassium-sparing diuretics within 28 days prior to day 0, or        during the study;    -   j. Use of tricyclic antidepressants or monoamine oxidase        inhibitor medications within 14 days prior to day 0, or during        the study.

Efficacy Assessments: Assessments include (a) abdominal subcutaneousadipose tissue layer thickness using 2-dimensional ultrasound; (b)abdominal skin fold thickness measurements using commercial pinchcalipers to measure the vertical skin folds directly over the separateinjection sites (2 cm to the right and left of the umbilicus); and (c)waist measurements taken at the level of the umbilicus/injection sites.

(a) 2-Dimensional Ultrasound

Procedure: Ultrasound assessment of the designated injection sites (2 cmto the right and left of the umbilicus) were undertaken at specifiedtime points. All study ultrasound recordings were performed using thesame model of ultrasound machine and the same probe (a 12 MHz linearprobe). Ultrasound gel was applied to the skin, and the thickness of thegel was sufficient to obtain a clear image without applying pressure tothe skin. To ensure valid capture data, the B-mode image was used to aidthe capture process to obtain good image quality. The ultrasoundtransducer was held vertical to the skin and using the skin markings forguidance, gently positioned in the sagittal plane with the mid-point ofthe transducer immediately above the marked injection site (bilateralsites on the right and left of the umbilicus). The transducer was heldvertical to the skin (at an angle incidence of 90°), theultrasonographer used digital calipers positioned in the center of thescan image and clicked on the pixel that marks the skin surface and thepixel represents the fat-fascial boundary. The distance between thepixels was recorded as the subcutaneous adipose tissue layer thickness.All images were produced by the same ultrasonographer. The change inmean subcutis thickness (in cm) from baseline as determined using2D-ultrasound was measured at 4 weeks, 5 weeks, and 8 weeks from thefirst day of treatment.

(b) Abdominal Skin Fold Measurement

Procedure:

Abdominal skin fold measurements utilizing a commercially availablepinch caliper were used per manufacturer instructions included with thecaliper device. Abdominal skin fold measurements were made by verticalpinch 2 cm lateral to the umbilicus at screening day and day 0 toconfirm subject eligibility. Abdominal skin fold measurements were alsoperformed throughout the study at specified times. Abdominal skin foldthickness measurements were taken directly over the separate injectionsites (2 cm to the right and left of the umbilicus). Measurements at theinjection sites were compared against baseline. Contra-lateral controlarea was also used to confirm that body fat remains relatively stableover the study duration.

(c) Waist Measurements

Procedure:

Measurements of waist were taken at specified time points. At day 0 whenthe injection sites were marked, the tip of the spinous process of thecorresponding lumbar vertebra at the level of the umbilicus were alsomarked. Waist measurements were taken at the level of the umbilicus inline with the two marked injection sites, and the posterior mark overthe lumbar vertebral spinous process. The full waist circumference, andthe right and the left hemi-circumferences were recorded.

Results:

FIG. 3A shows the mean change in full waist circumference from baselineover an 8 week period at specified time points in all patients receivingone injection per week of test drug for 4 weeks. FIG. 3B shows the meanchange in full waist circumference from baseline in these patients after8 weeks. FIGS. 3A and 3B demonstrate a dose-related treatment effectwith the group of patients receiving the 0.5 μg of salmeterol xinafoateand 1 μg of fluticasone propionate once per week for four weeksevidencing a change in waist circumference of about 3.5 cm at 8 weeks.Thus, the greatest change in full waist circumference was evidenced bythe patients receiving the least amount of active ingredient.

FIG. 4 demonstrates a weekly total dose-therapeutic efficacy responsecurve (based on change in waist circumference) for salmeterol xinafoate.The weekly dose with the greatest therapeutic efficacy was evidenced inpatients who received the lowest dose of 0.5 μg of salmeterol xinafoateat the lowest frequency (once per week for 4 weeks).

FIG. 8 demonstrates that the patients evidenced no significant change inmean weight during 8 weeks of study.

Safety Assessments:

Safety assessments were conducted at all time points. No serious adverseevents were observed in patients studied. Specifically, no adversecardiovascular events were observed, and no significant skin changes(e.g., atrophy, pigmentation, nodularity, or necrosis) were observed.Only minimal skin reactions (transient pain and irritation) wereobserved.

Example 3C Phase 2—a Double-Masked Evaluation of the Safety and Efficacyof Salmeterol Xinafoate and Fluticasone Propionate Co-Administered forSubcutaneous Injection for the Reduction of Subcutaneous AbdominalAdiposity

Objectives:

A Phase 2 study to examine the potential of selected doses of salmeterolxinafoate (SX) and fluticasone propionate (FP) co-administered to reduceabdominal subcutaneous adipose tissue, which is an interim surrogate toguide studies in subjects with symptomatic exophthalmos associated withthyroid-related eye disease (TED). The study also evaluated the safetyand efficacy of subcutaneous injections of salmeterol xinafoate (SX) andfluticasone propionate (FP) for the reduction of abdominal (peri- andinfra-umbilical) adiposity. The study compared the efficacy and safetyof salmeterol xinafoate (SX) and fluticasone propionate (FP) at 1.0 μgFP and 0.05 μg SX once weekly to 1 mL placebo once weekly for 8 weeksfor the reduction of measurable, abdominal subcutaneous adipose tissue.Additionally, the study evaluated in a double-masked, placebo-controlleddesign, the pharmacokinetics following multiple injected doses ofsalmeterol xinafoate (SX) and fluticasone propionate (FP) once weeklyfor 8 weeks. Other objectives of the study included exploration of anumber of secondary efficacy endpoints, and evaluation of potentialbiomarkers.

Study Design:

A double-masked, multiple-dose study of the safety and efficacy of0.05/1.0 or 0.5/1.0 μg/mL doses of salmeterol xinafoate (SX) andfluticasone propionate (FP) compared to placebo administered as twentytwo 1 mL regional subcutaneous injections once a week for 8 weeks in 60subjects with measurable abdominal subcutaneous adipose tissue.

Study Drug—Active Ingredient:

A 400 μg/mL sterile, preservative-free, clear, aqueous solution forinjection of SX is contained in a 5 mL single-use glass vial. Each 5 mLsingle-use glass vial contains 2.6 mL of SX solution (of 400 μg/mL). A25 mg/mL sterile, preservative free, clear, aqueous solution forinjection of FP is contained in a 2 mL single-use glass vial. Each 2 mLclear glass vial contains 1.0 mL of FP solution (25 mg/mL). Immediatelyprior to administration, a volume of SX (2.6 mL fill of 400 μg/mL in a5.0 mL vial) was mixed with FP (1.0 mL fill of 25 mg/mL in a 5.0 mLvial). The SX and FP should be stored frozen (−15° C. or below) andprotected from light until dose preparation. The mixed SX and FP shouldbe protected form light until administration. The SX and FP drugproducts are provided in nested drug packaging.

Placebo

Sterile saline, USP (0.9% sodium chloride) was used as the placebo.

The mixed SX and FP combination and placebo was administered astwenty-two 1 mL (for a total volume of 22 mL) into the abdominalsubcutaneous adipose tissue using a sterile, disposable, syringe with 27gauge and ⅜ inch needle.

Treatment Groups:

Eligible subjects in this multiple dose study were randomized in a 1:1:1ratio to receive twenty-two 1 mL subcutaneous injections of either 1.0μg/mL of FP+0.05 μg/mL SX or 1.0 μg/mL of FP+0.5 μg/mL SX or placebo.The twenty-two subcutaneous injections were spaced 4 cm apart andtreated a pre-marked midline abdominal area of adiposity that wasapproximately 16×14 cm². The sequence of once weekly subcutaneousinjections continued for 8 consecutive weeks for a total of 176injections administered into the marked abdominal area. Comprehensivesafety assessments were performed at each subject visit through the endof study on day 57+/−2 days.

Duration of Treatment:

The screening period lasted 30 days. The expected study duration was 9weeks: 8 weeks of a treatment period and 1 week post-therapy follow-upvisit for assessment of safety and efficacy.

Study Population:

Qualified male and female patients, 18 to 65 years of age, inclusive. Aqualified subject had a localized, measurable abdominal subcutaneousadipose tissue that was reported to be exercise-resistant anddiet-resistant.

Inclusion Criteria:

-   -   a. Male and female subjects, 18 to 55 years of age, having        provided informed written consent;    -   b. Subjects reporting dissatisfaction with their abdominal (peri        and infra-umbilical) subcutaneous adipose tissue, or who are        considering, or are in the process of seeking cosmetic reduction        of their abdominal adiposity;    -   c. BMI greater than or equal to 18.5 kg/m² and less than or        equal to 28 kg/m² and a history of stable weight in the last 3        months, and a variance of less than or equal to 5% between        screening at day 1.    -   d. History of a stable diet and exercise routine in the 3 months        prior to screening, and a willingness to adhere strictly to this        established routine during the period of study;    -   e. Female subjects who have a negative urine pregnancy test at        screening and day 1, and who agree to use adequate birth control        methods (abstinence, female partner, stabilized on oral        contraceptives for at least 2 months, implant, injection, IUD,        patch, NuvaRing®, condom and spermicidal, diaphragm and        spermicidal) throughout the study until completion of        post-treatment procedures for all.

Exclusion Criteria:

-   -   a. Females who are pregnant, lactating, and/or who are        childbearing but are not using adequate birth control methods;    -   b. Female subjects who are not within 12 months post-partum;    -   c. History of treatment of abdominal subcutaneous adipose tissue        including procedures (e.g. caesarean section, abdominoplasty,        liposuction), ablative contouring devices, mesotherapy or        lipolytic agents;    -   d. Subjects planning to embark on a weight loss or exercise        program during participation;    -   e. Subjects who partake of abdominal massaging and who are        unwilling to this therapy during the study;    -   f. Known hypersensitivity to the study drugs and/or any of their        components;    -   g. Prior or current enrollment in any Lithera study;    -   h. Concurrent enrollment in another investigational drug or        device study; or experimental or investigational drug or device        within 30 days, or for drugs times the elimination half-life        prior to day 1 if that is longer;    -   i. Any medical condition that in the opinion of the investigator        might jeopardize subject's safety or complicate study procedures        or assessments, including, but not limited to:        -   i. any bleeding, or connective tissue disorders;        -   ii. diabetes (Type I and II) or cardiovascular disease            (subjects with well-hypertension will not be excluded);        -   iii. history of major surgery within 30 days prior to day 1,            or planned surgery during the study period;        -   iv. any clinically-significant physical exam findings, as            determined by the investigator, at Screening or day 1;        -   v. lymphatic disease causing lymph edema, or other skin            conditions (e.g. eczema, tattoos, striae, keloids,            hypertrophic scars, or piercings) in the areas;        -   vi. abdominal asymmetry due to musculoskeletal            abnormalities, prior surgery, hernias or abdominal            organomegaly;        -   vii. history of any DSM-IV psychiatric disorder related to            body image (such anorexia nervosa, bulimia, body dysmorphic            disorder, etc.);        -   viii. any clinically-significant abnormal laboratory result            during Screening Day 1, as determined by the investigator.    -   j. Use of systemic, inhaled or topical corticosteroids, drugs        with anticoagulant activity (including chronic use of NSAIDs),        immunomodulators, anti-metabolites, β2-adrenergic receptor        agonists or β-adrenergic receptor blockers, nonpotassium-sparing        diuretics (e.g. loop or thiazide diuretics), or potent CYP 3A4        inhibitor drugs within 28 days prior to day 1, or during the        study;    -   k. Use of tricyclic antidepressants or monoamine oxidase        inhibitor medications within 14 days prior to day 1, or during        the study;    -   l. 12) Subjects unlikely or unable to comply with protocol        procedures or adhere to the study visit schedule.

Procedures:

All injections were performed in an outpatient setting. At each visit,subjects received a total of 22 subcutaneous injections (1 mL) toinfiltrate an area of approximately 16×14 cm². The study subjects hadblood collected for PK assessments at day 1, and day 50: on these dayssubjects remained at the clinic for 12 hours before being discharged.Once the study was completed and unmasked, only the serum samplescollected from subjects receiving the higher combination dose wereanalyzed. Subjects maintained their usual diet and exercise routineduring the study: any fat treatment, including but not limited toliposuction, mesotherapy and abdominal massaging was not allowed.Subjects underwent screening procedures at the Screening Visit. Thisvisit occurred within 30 days (day −30 to day 0) prior to studyrandomization at Day 1. Study procedures were explained to each subjectand written, informed consent was obtained prior to initiating anystudy-related procedures, including screening procedures. Qualifiedsubjects, who meet all Inclusion/Exclusion criteria, with baselinescreening laboratory tests results within normal limits as defined perprotocol, were scheduled for the Randomization Visit on Day 1. It wasrequired that all Randomization Visits (Day 1) be scheduled to ensurethat over the 8 week treatment period, weekly study drug administrationfor each subject occurs in a regular cycle, with doses of study drugadministered on the same day each week (±2 days).

Safety Assessments:

The following safety assessments were performed at the designated timepoints as specified in the protocol: (1) vital signs (systolic anddiastolic blood pressure, heart rate, breathing rate and bodytemperature); (2) clinical assessment of injection site reactions (localtolerability using the Injection Site Reaction Severity Scale); (3)clinical laboratory tests (hematology, serum chemistry, lipid panelincluding FFAs, and urine dipstick analysis); (4) and adverse events.Safety parameters monitored during this study were compared within eachtreatment group (i.e., changes from baseline) and between treatmentgroups.

Efficacy Assessments:

Efficacy assessments included: (1) circumferential measurements of theabdominal treatment area; (2) standardized digital images of thetreatment area to assess volumetric changes using a validatedmethodology and software; (3) Changes in the Patient Global Impressionof Severity, and the Patient Global Impression of Change questionsevaluating the overall treatment using ‘Before’ and ‘After’ photographs;(4) Changes in the Clinician Global Impression of Severity, and theClinician Global Impression of Change questions evaluating the overalltreatment using ‘Before’ and ‘After’ photographs (5) the AbdominalAppearance Questionnaire, a Patient Reported Outcome assessing changefrom baseline; and (6) caliper measurements of skin-fold thickness inthe abdominal treatment area.

PK Sample Collections:

A total of approximately 16 blood samples (5 mL each) were collected persubject during the study.

PK Parameters:

AUC 0-t, AUC 0-inf, C max, t max and t ½el.

Analytical Methods:

All SX and FP plasma samples were analyzed at the completion of thestudy using validated LC/MS/MS methods.

Study Endpoints:

The study endpoints included both safety assessments and evaluations ofefficacy. The primary efficacy endpoint was the change from baseline incircumferential abdominal measurements. Secondary endpoints included thechange from baseline in 3-D photographic assessments of volumetricchanges of the treatment area as assessed by a masked, central reader;skin-fold caliper measurements of the abdomen in the treatment area; andPatient and Clinician Global Impression of Severity questions, andPatient and Clinician Global Impression of Change questions; and theAbdominal Appearance Questionnaire, a patient-reported outcomeinstrument assessing the treatment response. ‘Before’ and ‘After’digital photographs (lateral and frontal) of the treatment areas takenat Baseline (day 1), at day 57±2 were used by the subjects in completingtheir Patient Global Impression of Change questions, and were also usedby the site PI who completed the Clinician Global Impression of Changequestions assessing each subject. A total of approximately 16 bloodsamples (5 mL each) were collected per subject during the study.

Results:

The results of this study show that the subcutaneous salmeterol andfluticasone formulations provide reductions in computer analyzed3-dimensional abdominal circumference and volume when administered onceweekly for 8 weeks. The 0.05 μg SX+1.0 μg FP treatment group (totalweekly SX/FP dose of 1.1 μg/22 μg) experienced the greatest reductionsin abdominal circumference (−1.23 cm vs. −0.1 cm for Placebo; p=0.048)and abdominal volume (−183 cc vs. +24 cc for Placebo; p=0.023) asassessed by the Canfield Vectra™ 3 D system. In this treatment group,32% of subjects lost >2 cm in abdominal circumference (vs. 5% forPlacebo). Consistent with greater lipolytic responsiveness of youngersubjects, a subgroup analysis showed that subjects less than 40 years ofage had greater abdominal circumference changes (−2.2 cm; p=0.004) andgreater volumetric changes (−360 cc; p=0.004) than subjects older than40. Similarly, a subgroup analysis showed that thinner subjects(skin-fold thickness less than 13.3 mm (study median)) had greaterabdominal circumference changes (−1.8 cm; F=0.053) and greatervolumetric changes (−309 cc; p=0.005) than subjects with more abdominalfat (skin-fold thickness less than 13.3 mm). Anterior abdominalflattening was observed in 2D images and was recognized as a reductionin grade of severity of the adiposity in treated subjects. Nosignificant change in weight was observed in any treatment group overthe 8-week trial. In the non-treatment study extension, reductions inabdominal circumference and volume in the 0.05 μg SX+1.0 μg FP treatmentgroup (total weekly SX/FP dose of 1.1 μg/22 μg) persisted for 12 weekspost-treatment. The dosage formulations were well-tolerated wheninjected weekly into the abdominal subcutaneous fat of healthy subjectswith the most commonly reported adverse events being mild, transientinjection site pain and irritation. There was no difference in injectionsite reactions between the subcutaneous salmeterol and fluticasoneformulations and Placebo-treated groups. There was no inflammation,nodularity or skin atrophy on physical examination of the treatmentarea. There were also no clinically significant changes in bloodpressure, heart rate, respiratory rate or temperature measurements.Plasma salmeterol and FP levels produced by the total weekly SX/FP doseof 11 μg/22 μg were ˜⅓-⅕ those of 505(b)(2) reference levels produced byan FDA-approved drug.

Example 4 Pharmaceutical Formulations

Subcutaneous Formulation

To prepare a parenteral pharmaceutical composition suitable forsubcutaneous administration, a salt of salmeterol, such as salmeterolxinafoate, is dissolved in PEG 400 which is stabilized with polysorbate80. Water is then added. This solution is stored in a single-use glassvial which is stored frozen and protected from light until dosepreparation. Fluticasone propionate is dissolved in PEG 400 which isalso stabilized by the addition of polysorbate 80. Water is then addedto the solution. This solution is stored in a single-use glass vialwhich is stored frozen and protected from light until dose preparation.The salmeterol and/or fluticasone propionate solutions are then dilutedto a suitable concentration for subcutaneous administration using adiluent made of an aqueous solution of 20% PEG 400, 1% polysorbate 80,and sterile water for injection.

Transcutaneous Formulation

Also provided herein is a transcutaneous formulation for administrationto a patient, and methods of treatment provided herein, including forexample methods for the reduction of adipose tissue, using thetranscutaneous formulations provided herein. In some embodiments, thetranscutaneous formulation contains about 0.01 to about 0.1% by weightfluticasone propionate, about 0.5% to about 10% by weight salmeterolxinafoate or formoterol fumarate, about 1% to about 75% propylene glycolor isopropyl alcohol, and optionally other excipients including but notlimited to transcutol, propyl gallate, water, and ethanol, wherein thetotal percent by weight is 100%.

The following transcutaneous formulations were topically administered tohuman cadaver skin ex vivo: (1) a transcutaneous formulation comprisingfluticasone propionate is in an amount of about 0.05% by weight,salmeterol xinafoate is in an amount of about 1% by weight, propyleneglycol is in an amount of about 5% by weight, transcutol is in an amountof about 15% by weight, propyl gallate is in an amount of about 0.025%by weight, water is in an amount of about 15% by weight, and ethanol isin an amount of about 64% by weight; and (2) a transcutaneousformulation comprising about 0.05% by weight fluticasone propionate, 1%by weight formoterol fumarate, propylene glycol in an amount of about50% by weight, and isopropyl alcohol in an amount of about 48.95% byweight.

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes are included withinthe spirit and purview of this application and scope of the appendedclaims. All publications, patents, and patent applications cited hereinare hereby incorporated by reference for all purposes.

What is claimed is:
 1. An aqueous formulation comprising a non-sustainedrelease formulation suitable for subcutaneous injection that comprises:(a) an adipose tissue-reducing lipophilic long-acting selective beta-2adrenergic receptor agonist or a salt thereof, that comprises a dose ofup to about 2 micrograms of the agonist and that is suitable forsubcutaneous injection; and (b) at least one subcutaneously acceptableinactive ingredient.
 2. The formulation of claim 1 wherein theformulation is formulated as a dose of up to about 1 microgram of thebeta-2 adrenergic receptor agonist.
 3. The formulation of claim 1wherein the formulation is formulated as a dose of up to about 500nanograms of the beta-2 adrenergic receptor agonist.
 4. The formulationof claim 1 wherein the formulation is formulated as a dose of up toabout 20 nanograms of the beta-2 adrenergic receptor agonist.
 5. Theformulation of claim 1 wherein the formulation is formulated to beadministered using a single needle, multiple needles, or a needlelessinjection device.
 6. The formulation of claim 1 wherein the dose of thebeta-2 adrenergic receptor agonist is formulated to be administeredthrough at least 2 sub-doses.
 7. The formulation of claim 1 wherein thedose of the beta-2 adrenergic receptor agonist is formulated to beadministered through at least 10 sub-doses.
 8. The formulation of claim1 wherein the lipophilic long-acting selective beta-2 adrenergicreceptor agonist is salmeterol xinafoate.
 9. The formulation of claim 8wherein the dose of salmeterol xinafoate is formulated to beadministered through at least 2 sub-doses.
 10. The formulation of claim9 wherein a single sub-dose comprises about 20 nanograms of salmeterolxinafoate.
 11. A method of treating a patient who has excess adiposetissue or reducing adipose tissue in a patient comprising subcutaneouslyadministering to said patient a non-sustained release pharmaceuticalformulation that is suitable for subcutaneous injection, the formulationcomprising: (a) an adipose tissue-reducing lipophilic long-actingselective beta-2 adrenergic receptor agonist or a salt thereof, thatcomprises a dose of up to about 2 micrograms of the agonist; and (b) atleast one subcutaneously acceptable inactive ingredient.
 12. The methodof claim 11 wherein the formulation is administered as a dose of up toabout 1 microgram of the beta-2 adrenergic receptor agonist.
 13. Themethod of claim 11 wherein the formulation is administered as a dose ofup to about 500 nanograms of the beta-2 adrenergic receptor agonist. 14.The method of claim 11 wherein the dose of the beta-2 adrenergicreceptor agonist is administered through at least 2 sub-doses.
 15. Themethod of claim 11 wherein the lipophilic long-acting selective beta-2adrenergic receptor agonist is salmeterol xinafoate.
 16. The method ofclaim 15 wherein the dose of salmeterol xinafoate is administeredthrough at least 10 sub-doses.
 17. The method of claim 16 wherein asingle sub-dose comprises up to about 50 nanograms of salmeterolxinafoate.
 18. The method of claim 11, wherein the method is a method oftreating a patient who has excess adipose tissue.
 19. The method ofclaim 11, wherein the method is a method of reducing adipose tissue in apatient.